Anti-Cancer SERCA Inhibitors Targeting Sorafenib-Resistant Human Papillary Thyroid Carcinoma.

Thyroid cancer is generally curable and, in many cases, can be completely treated, although it can sometimes recur after cancer therapy. Papillary thyroid cancer (PTC) is known as one of the most general subtypes of thyroid cancer, which take up nearly 80% of whole thyroid cancer. However, PTC may develop anti-cancer drug resistance via metastasis or recurrence, making it practically incurable. In this study, we propose a clinical approach that identifies novel candidates based on target identification and validation of numerous survival-involved genes in human sorafenib-sensitive and -resistant PTC. Consequently, we recognized a sarco/endoplasmic reticulum calcium ATPase (SERCA) in human sorafenib-resistant PTC cells. Based on the present results, we detected novel SERCA inhibitor candidates 24 and 31 via virtual screening. These SERCA inhibitors showed remarkable tumor shrinkage in the sorafenib-resistant human PTC xenograft tumor model. These consequences would be clinically worthwhile for the development of a new combinatorial strategy that effectively targets incredibly refractory cancer cells, such as cancer stem cells and anti-cancer drug-resistant cells.

Effects of Anti-Cancer Drug Sensitivity-Related Genetic Differences on Therapeutic Approaches in Refractory Papillary Thyroid Cancer.

Thyroid cancer (TC) includes tumors of follicular cells; it ranges from well differentiated TC (WDTC) with generally favorable prognosis to clinically aggressive poorly differentiated TC (PDTC) and undifferentiated TC (UTC). Papillary thyroid cancer (PTC) is a WDTC and the most common type of thyroid cancer that comprises almost 70-80% of all TC. PTC can present as a solid, cystic, or uneven mass that originates from normal thyroid tissue. Prognosis of PTC is excellent, with an overall 10-year survival rate >90%. However, more than 30% of patients with PTC advance to recurrence or metastasis despite anti-cancer therapy; consequently, systemic therapy is limited, which necessitates expansion of improved clinical approaches. We strived to elucidate genetic distinctions due to patient-derived anti-cancer drug-sensitive or -resistant PTC, which can support in progress novel therapies. Patients with histologically proven PTC were evaluated. PTC cells were gained from drug-sensitive and -resistant patients and were compared using mRNA-Seq. We aimed to assess the in vitro and in vivo synergistic anti-cancer effects of a novel combination therapy in patient-derived refractory PTC. This combination therapy acts synergistically to promote tumor suppression compared with either agent alone. Therefore, genetically altered combination therapy might be a novel therapeutic approach for refractory PTC.

Synergistic Anticancer Activity of N-Hydroxy-7-(2-Naphthylthio) Heptanomide, Sorafenib, and Radiation Therapy in Patient-Derived Anaplastic Thyroid Cancer Models.

Anaplastic thyroid cancer (ATC) is an undifferentiated and advanced form of thyroid cancer, accompanied with a high ratio of epigenetic adjustment, which occurs more than genetic mutations. In this study, we aimed to evaluate the synergistic anticancer effect (in vitro and in vivo) of the new combination of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) and sorafenib with radiation therapy in pre-clinical models of ATC. The ATC cell lines, YUMC-A1 and YUMC-A2, were isolated from the current patients who were treated with HNHA and sorafenib, either as monotherapy or combination therapy. Synergistic anticancer effect of the combination therapy on the intracellular signaling pathways and cell cycle was assessed via flow cytometry and immunoblot analysis. To examine tumor shrinkage activity in vivo, an ATC cell line-derived mouse xenograft model was used. Results showed that the combination therapy of HNHA and sorafenib with radiation promoted tumor suppression via caspase cleavage and cell cycle arrest in patient-derived ATC. In addition, the combination therapy of HNHA and sorafenib with radiation was more effective against ATC than therapy with HNHA or sorafenib with radiation. Thus, the combination of HNHA and sorafenib with radiation may be used as a novel curative approach for the treatment of ATC.

Long-term survival of patients with anaplastic thyroid cancer after multimodal treatment.

BACKGROUND: Anaplastic thyroid cancer (ATC) is among the most aggressive human malignancies, with a mean survival time of 6 months regardless of the treatment. METHODS: This retrospective study used the single-centre database system of the Gangnam Severance Hospital. The management and outcome data of 23 patients with a definitive histological diagnosis of ATC were reviewed. RESULTS: The 23 long-term survivors were 11 men and 12 women, with a mean age of 58 years. Nine patients had distant metastases at the time of diagnosis. Surgical debulking or complete resection of the tumour was performed for 19 patients, and chemotherapy was administered to 15 patients, radiotherapy to 18 patients, and tyrosine kinase inhibitors to 6 patients. In total, 14 patients were treated with a combination of surgery and radiotherapy with or without chemotherapy. Only 5 patients were treated with surgery alone. Overall, 15 patients underwent R0 resection, 2 underwent R1 resection, and 2 underwent R2 resection. The median survival was 1,090 days, the median follow-up was 646 days, and the 2- and 3-year survival rates were 59.7% and 35.8%, respectively. A total of 10 patients died: 7 with local disease and 3 with distant metastasis. CONCLUSIONS: Although ATC is typically an incurable disease, patients with ATC who underwent multimodality treatments including resection, chemotherapy, radiotherapy, and thyrosine kinase inhibitors would survive more than 1 year.

Amelioration of amyloid ß-FcγRIIb neurotoxicity and tau pathologies by targeting LYN.

SRC-family kinases (SFKs) have been implicated in Alzheimer's disease (AD), but their mode of action was scarcely understood. Here, we show that LYN plays an essential role in amyloid ß (Aß)-triggered neurotoxicity and tau hyperphosphorylation by phosphorylating Fcγ receptor IIb2 (FcγRIIb2). We found that enzyme activity of LYN was increased in the brain of AD patients and was promoted in neuronal cells exposed to Aß 1-42 (Aß1-42). Knockdown of LYN expression inhibited Aß1-42-induced neuronal cell death. Of note, LYN interacted with FcγRIIb2 upon exposure to Aß1-42 and phosphorylated FcγRIIb2 at Tyr273 within immunoreceptor tyrosine-based inhibitory motif in neuronal cells. With the use of the structure-based drug design, we isolated KICG2576, an ATP-competitive inhibitor of LYN. Determination of cocrystal structure illustrated that KICG2576 bound to the cleft in the LYN kinase domain and inhibited LYN with a half-maximal inhibitory concentration value of 0.15 µM. KICG2576 inhibited Aß- or FcγRIIb2-induced cell death, and this effect was better than pyrazolopyrimidine 1, a widely used inhibitor of SFK. Upon exposure to Aß, KICG2576 blocked the phosphorylation of FcγRIIb2 and translocation of phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, a binding protein to the phosphorylated FcγRIIb2, to the plasma membrane, resulting in the inhibition of tau hyperphosphorylation, the downstream event of Aß1-42-FcγRIIb2 binding. Furthermore, intracerebroventricular injection of KICG2576 into mice ameliorated Aß-induced memory impairment. These results suggest that LYN plays a crucial role in Aß1-42-mediated neurotoxicity and tau pathology, providing a therapeutic potential of LYN in AD.-Gwon, Y., Kim, S.-H., Kim, H. T., Kam, T.-I., Park, J., Lim, B., Cha, H., Chang, H.-J., Hong, Y. R., Jung, Y.-K. Amelioration of amyloid ß-FcγRIIb neurotoxicity and tau pathologies by targeting LYN.

SoLAT (Sorafenib Lenvatinib alternating treatment): a new treatment protocol with alternating Sorafenib and Lenvatinib for refractory thyroid Cancer.

BACKGROUND: In the last decade, several tyrosine kinase inhibitors (TKIs), which disrupt pathways involved in the proliferation and tumorigenesis of thyroid cancer, have been extensively studied. Two different TKIs, lenvatinib and sorafenib, were recently approved by both the US FDA and European Medicine Agency. Until date, the duration of the TKI response is not sufficient and resistance eventually occurs. The goal of this study was to investigate a new treatment protocol, SoLAT, using sorafenib and lenvatinib alternatively on refractory thyroid cancer. METHODS: Patient-derived aggressive papillary thyroid cancer (PTC) cell lines from patients with biochemical and histologically proven aggressive RAI-refractory papillary thyroid cancer were exposed to sorafenib and lenvatinib alternatively. Human thyroid cancer cell xenografts were obtained by injecting patient-derived aggressive PTC cell lines into the flank of female BALB/c nude mice. Tumor-bearing mice were treated with sorafenib and lenvatinib alternatively. Cell viability assay, immunofluorescence analysis, confocal imaging, immunoblot analysis, flow cytometry analysis of cell cycle and a tube formation assay were performed. RESULTS: SoLAT was more effective for advanced PTC cell lines than individual treatment. Immunoblot analysis showed that SoLAT markedly increased levels of cell cycle inhibitors (p53 and p21), and pro-apoptotic factors (Apaf-1 and cleaved caspase 3) and decreased levels of positive cell cycle regulators (cyclin D1, CDK4, CDK6) and anti-apoptotic factors (p-NFκB, Bcl-2). Increased sub-G0/G1 population was observed in the SoLAT group, leading to apoptosis, cell cycle arrest, and strong inhibition of advanced PTC cell viability. SoLAT reduced the level of EMT markers such as vimentin, E-cadherin, Snail and Zeb1 by FGFR inhibition. In the xenograft model, individual treatment with sorafenib or lenvatinib did not markedly suppress patient-derived aggressive PTC cell xenograft tumors, whereas SoLAT significantly suppressed the proliferation of these tumors. CONCLUSIONS: SoLAT was more effective than individual treatment with sorafenib or lenvatinib in inhibiting PTC progression by inducing cell cycle arrest. Studies using both in vitro cell culture and an in vivo xenograft model provided evidence of tumor shrinkage with SoLAT. We suggest that these effects may be due to reduced EMT-mediated drug resistance in the aggressive PTC model.

Anti-cancer Effects of HNHA and Lenvatinib by the Suppression of EMT-Mediated Drug Resistance in Cancer Stem Cells.

Anaplastic thyroid cancer (ATC) constitutes less than 2% of total thyroid cancers but accounts for 20-40% of thyroid cancer-related deaths. Cancer stem cell drug resistance represents a primary factor hindering treatment. This study aimed to develop targeted agents against thyroid malignancy, focusing on individual and synergistic effects of HNHA (histone deacetylase), lenvatinib (FGFR), and sorafenib (tyrosine kinase) inhibitors. Patients with biochemically and histologically proven papillary thyroid cancer (PTC) and ATC were included. Cell samples were obtained from patients at the Thyroid Cancer Center, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. PTC and ATC cells were treated with lenvatinib or sorafenib, alone or in combination with HNHA. Tumor-bearing mice (10/group) were administered 10 mg/kg lenvatinib (p.o.) or 40 mg/kg sorafenib (p.o.), alone or in combination with 25 mg/kg HNHA (i.p.) once every three days. Gene expression in patient-derived PTC and ATC cells was compared using a microarray approach. Cellular apoptosis and proliferation were examined by immunohistochemistry and MTT assays. Tumor volume and cell properties were examined in the mouse xenograft model. HNHA-lenvatinib combined treatment induced markers of cell cycle arrest and apoptosis and suppressed anti-apoptosis markers, epithelial-mesenchymal transition (EMT), and the FGFR signaling pathway. Combined treatment induced significant tumor shrinkage in the xenograft model. HNHA-lenvatinib combination treatment thus blocked the FGFR signaling pathway, which is important for EMT. Treatment with HNHA-lenvatinib combination was more effective than either agent alone or sorafenib-HNHA combination. These findings have implications for ATC treatment by preventing drug resistance in cancer stem cells.

Synergistic Activity of N-hydroxy-7-(2-naphthylthio) Heptanomide and Sorafenib Against Cancer Stem Cells, Anaplastic Thyroid Cancer.

Anaplastic thyroid carcinoma (ATC) although rare is the most deadly form of thyroid cancer. The fatality rate for ATC is high-pitched, the survival rate at 1 year after diagnosis is <20%. Control of ATC is severely hard and widespread with unpredictability. We Previous proved that histone gene reviser and epigenetic changes role significant parts in papillary and anaplastic thyroid cancer tumorigenesis. Herein, the goal of this study was to investigate the anti-tumor activities of a HDAC inhibitor, HNHA alone and in combination with sorafenib in ATC cells in vitro and in vivo and to explore its effects on apoptotic cell death pathways. Three ATC cell lines were exposed to sorafenib in the presence or absence of HNHA, and cell viability was determined by MTT assay. Effects of combined treatment on cell cycle and intracellular signaling pathways were assessed by flow cytometry and western blot analysis. The ATC cell lines xenograft model was used to examine the anti-tumor activity in vivo. Our data showed that HNHA and sorafenib synergistically decreased cell viability in ATC cells, and also significantly increased apoptotic cell death in these cells, as proved by the cleavage of caspase-3 and DNA fragmentation. HNHA and sorafenib combination was reduced anti-apoptotic factor in ATC. Thus, combination therapy with HNHA and sorafenib significantly decreased vessel density, and most significantly reduced tumor volume and increased survival in ATC xenografts. These results propose that HNHA in combination with sorafenib has significant anti-cancer activity in preclinical models, potentially suggesting a new clinical approach for patients of advanced thyroid cancer type.

Thyroidectomy without lateral neck dissection for papillary thyroid carcinoma with lateral neck lymph node metastases and negative intraoperative frozen section.

BACKGROUND: The purpose of this study was to investigate the outcomes of patients with papillary thyroid cancer (PTC) with lateral neck metastasis according to their permanent pathology report but negative frozen section findings who did not undergo lateral neck dissection. METHODS: Between September 2009 and December 2011, 575 patients at Gangnam Severance Hospital (Seoul, Korea) underwent frozen section analysis for a suspicious lateral neck lymph node. In 16 patients, the intraoperative findings were negative, but lateral neck metastasis was diagnosed on the basis of permanent pathology findings. The outcomes of these patients who underwent thyroidectomy but not lateral neck dissection were retrospectively investigated. RESULTS: One patient underwent a subsequent lateral neck dissection. After a mean (SD) follow-up period of 42.1 (8.5) months, none of the patients had distant metastasis. CONCLUSION: Total thyroidectomy with subsequent lateral neck dissection is not necessary in patients with PTC who are diagnosed with lateral neck metastasis according to their permanent pathology report but have negative intraoperative frozen section findings.

Tuberculosis cervical lymphadenopathy mimics lateral neck metastasis from papillary thyroid carcinoma.

BACKGROUND: Tuberculosis (TB) lymphadenitis is a frequent cause of lymphadenopathy in areas in which TB is endemic. Cervical lymphadenopathy in TB can mimic lateral neck metastasis (LNM) from papillary thyroid carcinoma (PTC). This study evaluated the clinicopathological features of patients with PTC and TB lateral neck lymphadenopathy. METHODS: Of the 9098 thyroid cancer patients who underwent thyroid cancer surgery at the Thyroid Cancer Center of Gangnam Severance Hospital between January 2009 and April 2013, 28 had PTC and showed TB lymphadenopathy of the lateral neck node. The clinicopathological features of these 28 patients were evaluated. RESULTS: Preoperatively, all 28 patients were diagnosed with PTC and showed cervical lymphadenopathy. All had radiological characteristics suspicious of metastasis in lateral neck nodes. Based upon the results from intraoperative frozen sections, lymph node dissection (LND) was not performed on 19 patients. Seven of eight patients who underwent LND had metastasis combined with tuberculous lymphadenopathy, with the remaining patient negative for LNM. CONCLUSIONS: Intraoperative sampling and frozen sectioning of lymph nodes suspicious of metastasis can help avoid unnecessary LND for tuberculous lymphadenopathy.

Potential anti-cancer effect of N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), a novel histone deacetylase inhibitor, for the treatment of thyroid cancer.

BACKGROUND: Thyroid cancer has been indicated to have a higher global proportion of DNA methylation and a decreased level of histone acetylation. Previous studies showed that histone gene reviser and epigenetic changes role significant parts in papillary and anaplastic thyroid cancer tumorigenesis. The goal of this research was to study the endoplasmic reticulum (ER) stress-mediated actions of the dominant histone deacetylase (HDAC) inhibitor, N-hydroxy-7-(2-naphthylthio) hepatonomide (HNHA), in thyroid cancer and to explore its effects on apoptotic cell death pathways. METHODS: Experiments were achieved to conclude the effects of HNHA in papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) cell lines and xenografts, as compared with two other established HDAC inhibitors (SAHA; suberoylanilide hydroxamic acid and TSA; trichostatin A). RESULTS: Apoptosis, which was induced by all HDAC inhibitors, was particularly significant in HNHA-treated cells, where noticeable B-cell lymphoma-2 (Bcl-2) suppression and caspase activation were observed both in vitro and in vivo. HNHA increased Ca(2+) release from the ER to the cytoplasm. ER stress-dependent apoptosis was induced by HNHA, suggesting that it induced caspase-dependent apoptotic cell death in PTC and ATC. PTC and ATC xenograft studies demonstrated that the antitumor and pro-apoptotic effects of HNHA were greater than those of the established HDAC inhibitors. These HNHA activities reflected its induction of caspase-dependent and ER stress-dependent apoptosis on thyroid cancer cells. CONCLUSIONS: The present study indicated that HNHA possibly provide a new clinical approach to thyroid cancers, including ATC.

Recovery from Permanent Hypoparathyroidism After Total Thyroidectomy.

BACKGROUND: Permanent hypoparathyroidism after total thyroidectomy is a rare but potentially serious iatrogenic complication. The aim of this study was to investigate the rate of recovery from postoperative, permanent hypoparathyroidism in patients undergoing thyroidectomy without parathyroid autotransplantation. METHODS: This study was a prospective case series with a postoperative follow-up of up to 3 years. We enrolled patients with thyroid cancer who underwent total thyroidectomy with central compartment dissection, with or without lateral neck dissection, and who had postoperative permanent hypoparathyroidism, defined as serum levels of intact parathyroid hormone (PTH) <15 pg/mL for at least 1 year. In the postoperative follow-up period, the serum levels of PTH and calcium were measured regularly. Recovery from permanent hypoparathyroidism was defined as return to normal serum levels of PTH (15-65 pg/mL) and calcium (8.5-10.1 mg/dL) without calcium and/or vitamin D supplementation. RESULTS: In the 1467 patients who underwent total thyroidectomy, 22 presented with permanent postoperative hypoparathyroidism. In 5 of these 22 patients, the PTH levels increased steadily and returned to normal in 27.6±2.9 months, after which supplementation of calcium and vitamin D could be discontinued. CONCLUSIONS: Although recovery from permanent hypoparathyroidism is rare, patients should be monitored for serum PTH levels so that unnecessary treatments such as calcium and vitamin D supplementation can be avoided.

An evaluation of the effectiveness of hyaluronidase in the selective nerve root block of radiculopathy: a double blind, controlled clinical trial.

STUDY DESIGN: Prospective, double-blind, randomized controlled trial. PURPOSE: To determine the ability of hyaluronidase to provide longer lasting pain relief and functional improvement in patients with lumbar radiculopathy. OVERVIEW OF LITERATURE: Selective nerve root block (SNRB) is a good treatment option in lumbar radiculopathy. We studied the effectiveness of hyaluronidase when added to the traditional SNRB regimen. METHODS: A sample size of 126 patients per group was necessary. A sample of 252 patients who underwent an injection procedure with or without hyaluronidase due to radiculopathy was included in this study. The patients were randomly divided into two groups: the control (C) group and the hyaluronidase (H) group. After SNRB due to radiculopathy, the visual analog scale (VAS) was compared at 2, 4, 6, 8, and 12 weeks between the two groups, and the Oswestry disability index (ODI) was compared at 12 weeks between the two groups. RESULTS: Both groups seemed to have general improvement in VAS, but in C group, the VAS was higher than the H group 2 and 4 weeks after the surgery, and the difference in time-group change between 2 groups was statistically significant (p <0.05). ODI improved in both groups, and the difference in time-group change between 2 groups was not statistically significant (p >0.05). CONCLUSIONS: The rebound pain (the re-occurrence of pain within 2-4 weeks after injection) that occurs within 2-4 weeks after the injection of the routine regimen can be reduced when hyaluronidase is added to the routine SNRB regimen.

Reducing neck incision length during thyroid surgery does not improve satisfaction in patients.

Postoperative neck cosmesis is a major concern of patients undergoing thyroid surgery. Patients will likely be more satisfied with the long-term cosmetic appearance of smaller than larger thyroidectomy scars. We, therefore, investigated the relationship between scar length following conventional thyroid surgery and patient satisfaction. An anonymous scar-assessment questionnaire was administered to patients who underwent conventional thyroid surgery. The 2,041 patients were asked to rate their satisfaction with their scars on a ten-point Likert scale, with one being very unsatisfied and ten being very satisfied. The mean satisfaction score was significantly lower in the benign condition than in malignancy (6.9 ± 2.5 vs. 7.4 ± 2.5; p = 0.021), whereas there were no differences in satisfaction score among subgroups of patients with benign condition (p = 0.837). In patients with thyroid cancer, the mean satisfaction scores were similar among subgroups according to operation type and scar length (p = 0.820). Incision length was not associated with patient satisfaction in thyroid surgery patients and therefore may not be critical in decision making for thyroid cancer surgery.

Solitary lateral neck node metastasis in papillary thyroid carcinoma.

BACKGROUND: Papillary thyroid carcinoma (PTC) is associated with a high incidence of regional node metastasis, but the patterns of lateral neck node metastasis (LNM) vary. Occasionally, a solitary LNM (SLNM) is seen in PTC patients. We therefore assessed whether selective single level node dissection is appropriate in PTC patients with SLNM. METHODS: We retrospectively reviewed the medical records of 241 PTC patients who underwent total thyroidectomy with central neck dissection plus ipsilateral internal jugular node dissection (level II to IV) between January 2010 and December 2011. Of these patients, 51 had SLNM and 190 had multiple LNM (MLNM). The clinicopathologic characteristics of the two groups were compared. RESULTS: Age, gender ratio, and numbers of lateral neck nodes harvested (29.4±11.0 versus 30.3±9.5; P=0.574) were similar in the SLNM and MLNM groups. Mean primary tumor size was significantly smaller in the SLNM than in the MNLM group (1.03 cm versus 1.35 cm; P=0.037). The proportion of patients with primary tumor≤1 cm was significantly greater in the SLNM group (60.8% versus 38.4%; P=0.006), whereas the proportion with maximal node size≤0.7 cm (28.9% versus 73.3%; P<0.001) and the proportion with capsular invasion (62.7% versus 83.7%, P=0.002) were significantly lower in the SLNM than in the MLNM group. CONCLUSIONS: Selective single level neck dissection can be considered as an alternative to systemic lateral neck dissection in PTC patients with SLNM, maximal metastatic node size≤0.7 cm, and no extrathyroidal invasion.

Design, synthesis and biological evaluation of novel imidazopyridines as potential antidiabetic GSK3ß inhibitors.

Design, synthesis and biological evaluation of the imidazopyridine analogs as novel GSK3ß inhibitors for treatment of type 2 diabetes mellitus are described. Most of the analogs exhibited excellent inhibitory activities (IC50<44 nM) against glycogen synthase kinase 3ß (GSK3ß). The structure-activity relationship (SAR) of the imidazopyridine analogs and the binding mode of analog 23 in the catalytic domain of GSK3ß, based on our X-ray crystallography study, are described. In particular, analog 28, which was selected as a potential drug candidate for treatment of type 2 diabetes mellitus, exhibited excellent GSK3ß inhibition, pharmacokinetic profiles and blood glucose lowering effect in mouse.

Molecular mechanism of activation of human Cdc7 kinase: bipartite interaction with Dbf4/activator of S phase kinase (ASK) activation subunit stimulates ATP binding and substrate recognition.

Cdc7 is a serine/threonine kinase conserved from yeasts to human and is known to play a key role in the regulation of initiation at each replication origin. Its catalytic function is activated via association with the activation subunit Dbf4/activator of S phase kinase (ASK). It is known that two conserved motifs of Dbf4/ASK are involved in binding to Cdc7, and both are required for maximum activation of Cdc7 kinase. Cdc7 kinases possess unique kinase insert sequences (kinase insert I-III) that are inserted at defined locations among the conserved kinase domains. However, precise mechanisms of Cdc7 kinase activation are largely unknown. We have identified two segments on Cdc7, DAM-1 (Dbf4/ASK interacting motif-1; amino acids 448-457 near the N terminus of kinase insert III) and DAM-2 (C-terminal 10-amino acid segment), that interact with motif-M and motif-C of ASK, respectively, and are essential for kinase activation by ASK. The C-terminal 143-amino acid polypeptide (432-574) containing DAM-1 and DAM-2 can interact with Dbf4/ASK. Characterization of the purified ASK-free Cdc7 and Cdc7-ASK complex shows that ATP binding of the Cdc7 catalytic subunit requires Dbf4/ASK. However, the "minimum" Cdc7, lacking the entire kinase insert II and half of kinase insert III, binds to ATP and shows autophosphorylation activity in the absence of ASK. However, ASK is still required for phosphorylation of exogenous substrates by the minimum Cdc7. These results indicate bipartite interaction between Cdc7 and Dbf4/ASK subunits facilitates ATP binding and substrate recognition by the Cdc7 kinase.

Structural basis of triclosan resistance.

Triclosan (5-chloro-2-(2,4-dichloro-phenoxy)-phenol, TCL) is a well known inhibitor against enoyl-acyl carrier protein reductase (ENR), an enzyme critical for cell-wall synthesis of bacteria. The inhibitory concentration at 50% inhibition (IC(50)) of TCL against the Escherichia coli ENR is 150nM for wild type (WT), 380, 470 and 68,500nM for Ala, Ser and Val mutants, respectively. To understand this high TCL resistance in the G93V mutant, we obtained the crystal structures of mutated ENRs complexed with TCL and NAD(+). The X-ray structural analysis along with the ab initio calculations and molecular dynamics simulations explains the serious consequence in the G93V mutant complex. The major interactions around TCL due to the aromatic(cation)-aromatic and hydrogen bonding interactions are found to be conserved both in WT and mutant complexes. Thus, the overall structural change of protein is minimal except that a flexible α-helical turn around TCL is slightly pushed away due to the presence of the bulky valine group. However, TCL shows substantial edge-to-face aromatic (π)-interactions with both the flexible R192-F203 region and the residues in the close vicinity of G93. The weakening of some edge-to-face aromatic interactions around TCL in the G93V mutant results in serious resistance to TCL. This understanding is beneficial to design new generation of antibiotics which will effectively act on the mutant ENRs.

Clinical characteristics of ischemic colitis according to location.

PURPOSE: The aim of this study was to analyze various clinical characteristics of ischemic colitis according to its location. METHODS: The medical records of 92 cases of gastrointestinal ischemic colitis (IC) diagnosed at Bundang CHA Hospital from 1995 to 2008 were reviewed and analyzed retrospectively. The patients were diagnosed by using colonoscopic biopsies or laparotomy findings. The patients were divided into two groups, right and left, according to the main involvement area of the IC at the embryologic boundary line of the distal transverse colon, and the two groups were compared as to clinical characteristics and co-morbid diseases. RESULTS: Left IC was present in 59 patients (64.1%) and right IC in 33 patients (35.9%). No differences between the two groups in terms of clinical characteristics, cardiovascular disease and diabetes mellitus were observed. However, in 16 cases with renal failure, 10 patient had right IC and 6 patients had left IC, and this difference had statistical significance (P = 0.014). Among the 16, the 11 patients requiring hemodialysis included 8 with right IC (24.2%) and 3 with left IC (5.1%; P = 0.009). Among the 19 cases of severe IC requiring surgical treatment or involving mortality, irrespective of surgery, 11 patients showed right IC and 8 patients showed left IC (P = 0.024). CONCLUSION: Right-side ischemic colitis was significantly associated with renal failure and disease severity, so patients with right-side colon ischemia should be more carefully observed and managed.

Estrone and estradiol C-16 derivatives as inhibitors of type 1 17beta-hydroxysteroid dehydrogenase.

Three series of steroid derivatives, enones 1, enols 2 and saturated alcohols 3, were easily synthesized from estrone according to a sequence of three reactions: an aldol condensation with an aromatic aldehyde (R(a-g)CHO) to afford 1, the carbonyl reduction of 1 to obtain the enol 2, and the double bond reduction of 2 to give 3 with the R(a-g) group 16beta-oriented. All compounds were tested as inhibitors of type 1 17beta-HSD. The inhibitory potency increases in the following order 1<2<3, suggesting that the presence of a flexible 16beta-methylene group allows a better positioning of the aryl moiety. With an IC50 of 0.8 microM, the 16beta-benzyl-E2 (3a) is the best inhibitor in this series.