Anti-diabetic effects of Protaetia brevitarsis in pancreatic islets and a murine diabetic model.

OBJECTIVE: In this study, the antidiabetic efficacy of Protaetia brevitarsis in alloxan-treated pancreatic islets and db/db mice was investigated. P. brevitarsis was tested for alloxan-mediated cytotoxicity and nitric oxide production in mice pancreatic islets. MATERIALS AND METHODS: The anti-diabetic effect of P. brevitarsis was also evaluated in db/db mice after 4 weeks of administration. Biochemical analysis, oral glucose tolerance test (OGTT), and pancreatic histological analysis were performed. RESULTS: P. brevitarsis displayed hypoglycemic activity in alloxan-treated mice pancreatic islets. Our results showed that P. brevitarsis protects pancreatic islets from cytotoxicity. Moreover, daily oral supplementation with P. brevitarsis for 4 weeks reduced plasma glucose levels without affecting body weight and food intake, elevated glucose tolerance in OGTT, improved blood lipid parameters, inhibited fat accumulation, and restored islet structure of db/db mice. CONCLUSIONS: The present study provided evidence for the anti­diabetic effect of P. brevitarsis in alloxan-treated pancreatic islets and db/db mice. These results suggest that P. brevitarsis may be used as an adjunctive anti-diabetic agent or as a functional food.

Enhanced antimicrobial stewardship based on rapid phenotypic antimicrobial susceptibility testing for bacteraemia in patients with haematological malignancies: a randomized controlled trial.

OBJECTIVES: Recently, rapid phenotypic antimicrobial susceptibility testing (AST) based on microscopic imaging analysis has been developed. The aim of this study was to determine whether implementation of antimicrobial stewardship programmes (ASP) based on rapid phenotypic AST can increase the proportion of patients with haematological malignancies who receive optimal targeted antibiotics during early periods of bacteraemia. METHODS: This randomized controlled trial enrolled patients with haematological malignancies and at least one positive blood culture. Patients were randomly assigned 1:1 to conventional (n = 60) or rapid phenotypic (n = 56) AST. The primary outcome was the proportion of patients receiving optimal targeted antibiotics 72 hr after blood collection for culture. RESULTS: The percentage receiving optimal targeted antibiotics at 72 hr was significantly higher in the rapid phenotypic AST group (45/56, 80.4%) than in conventional AST group (34/60, 56.7%) (relative risk (RR) 1.42, 95% confidence interval (CI) 1.09-1.83). The percentage receiving unnecessary broad-spectrum antibiotics at 72 hr was significantly lower (7/26, 12.5% vs 18/60, 30.0%; RR 0.42, 95% CI 0.19-0.92) and the mean time to optimal targeted antibiotic treatment was significantly shorter (38.1, standard deviation (SD) 38.2 vs 72.8, SD 93.0 hr; p < 0.001) in the rapid phenotypic AST group. The mean time from blood collection to the AST result was significantly shorter in the rapid phenotypic AST group (48.3, SD 17.6 vs 83.1, SD 22.2 hr). DISCUSSION: ASP based on rapid phenotypic AST can rapidly optimize antibiotic treatment for bacteraemia in patients with haematological malignancy. Rapid phenotypic AST can improve antimicrobial stewardship in immunocompromised patients.

Role of thymoglobulin in matched sibling allogeneic hematopoietic stem cell transplantation with busulfan and fludarabine conditioning in myeloid malignanicies.

In vivo T-cell depletion using anti-thymocyte globulin (ATG) is widely used in allogeneic hematopoietic stem cell transplantation (HSCT) for prophylaxis of GvHD. We investigated the influence of thymoglobulin dose (an ATG) on GvHD following matched sibling donor (MSD) HSCT with a busulfan and fludarabine preparative regimen. Medical records of 180 patients who received MSD HSCT with a conditioning regimen of busulfan, fludarabine, and ATG (BuFluATG) were reviewed retrospectively. The median age was 53 years (range 18-68). Initial diagnoses were acute myeloid leukemia (73.3%) and myelodysplastic syndrome (26.7%). Forty-four and 68 patients (24.4 and 37.7%) experienced acute and chronic GvHD of any grade, respectively. High-dose (⩾4.5 mg/kg) ATG was independently associated with decreased risk of acute GvHD (hazard ratio=0.36, 95% confidence interval (CI): 0.15-0.84, P=0.019) compared to low-dose ATG (<4.5 mg/kg). Although ATG dose was associated with the risk of acute GvHD, it was not associated with the risk of chronic GvHD in our study. A higher dose (⩾4.5 mg/kg) of ATG decreases the risk of acute GvHD but had no significant impact on disease-free survival in MSD HSCT patients conditioned with BuFluATG. The optimal dose of ATG should be further investigated in a large prospective study context.

Simultaneous Subtotal Pancreatectomy and Streptozotocin Injection for Diabetes Modeling in Cynomolgus Monkeys.

BACKGROUND: In an experimental animal model of islet transplantation, stable induction of insulin-dependent diabetes mellitus (IDDM) and islet isolation from donor pancreas are essential. Total pancreatectomy for IDDM induction and islet procurement in nonhuman primates leads to unwanted loss of exocrine function and may lead to morbidities associated with IDDM. METHODS: IDDM induction with streptozotocin (STZ) is associated with drug toxicity of STZ and necessitates the killing of another animal for islet procurement. In this study, we performed a subtotal pancreatectomy combined with reduced STZ injection to induce IDDM and procure islets in a nonhuman primate model. RESULTS: Twelve cynomolgus monkeys received low-dose STZ injections (60 mg/kg) simultaneously with subtotal pancreatectomy. All monkeys recovered from the procedure without complications. IDDM was induced in the animals. 57,691 ± 16,050 islets were isolated from the resected pancreas and transplanted into other monkeys. CONCLUSIONS: Simultaneous subtotal pancreatectomy and low-dose STZ injection represent an effective and safe method to create an animal model of insulin dependence diabetes, while at the same time providing sufficient amounts of fresh islet cells for allotransplantation without requiring killing of additional animals.

A phase II study of everolimus (RAD001), an mTOR inhibitor plus CHOP for newly diagnosed peripheral T-cell lymphomas.

BACKGROUND: Everolimus, an oral mTOR inhibitor, has single-agent activity against relapsed lymphomas. Thus, we carried out a phase II study of everolimus in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) as a first-line treatment for patients with peripheral T-cell lymphoma (PTCL) based on our phase I study results. PATIENTS AND METHODS: Participants (n = 30) received CHOP with 5 mg everolimus per day from day 1 to 14 every 21 days for a total of six cycles. The primary end point was the overall response rate (ORR), which included complete response (CR) and partial response (PR) to this regimen. Immunohistochemistry was used to evaluate the expression of phosphatase and tensin homology (PTEN) and phosphorylated S6 kinase (pS6K) as a response. RESULTS: The objective response rate was 90% with CR (n = 17) and PR (n = 10). The CR rate was different among subtypes; angioimmunoblastic T-cell lymphoma (AITL, n = 3) had a CR whereas PTCL-not-otherwise specified and ALK-negative anaplastic large-cell lymphoma (ALCL) patients showed 63% (12/19) and 29% (2/7) of CR rate, respectively. This difference in CR rate among subtypes was associated with PTEN loss because PTEN loss was not seen in AITL but 33% of ALCL patients. The most common toxicity was hematological, with 80% of patients experiencing at least one event of grade 3/4 neutropenia, and 60% of patients had grade 3/4 thrombocytopenia. CONCLUSION: The everolimus plus CHOP was effective for PTCL patients, and its efficacy might be related with the preservation of PTEN.

Relevance of TSH-receptor antibody levels in predicting disease course in Graves' orbitopathy: comparison of the third-generation TBII assay and Mc4-TSI bioassay.

AIMS: To investigate if TSH-receptor antibody (TRAb) levels measured in early Graves' orbitopathy (GO) stages are predictive of clinical disease course beyond 1 year after initial GO diagnosis and to compare performance of two newly developed TRAb assays (third-generation thyrotropin-binding inhibitor immunoglobulin (TBII) assay vs Mc4-thyroid-stimulating immunoglobulin (TSI) bioassay) in predicting disease course. METHODS: Newly diagnosed, untreated GO patients whose duration of ocular symptoms was less than 6 months were included. One year after initial diagnosis, all patients were classified as presenting either a mild (Group 1) or severe course (Group 2) according to their clinical manifestations. The measurements of two TRAb assays at initial GO diagnosis were used for analysis. RESULTS: Data from 112 patients were available for analysis. Seventy-three patients (65.2%) were designated as Group 1, and 39 patients (34.8%) as Group 2. Patients with higher initial TRAb levels demonstrated a higher risk of severe disease course upon multiple regression analysis (P<0.01). The cutoff values for the prediction of severe course of the third-generation TBII and Mc4-TSI assays were 10.67 IU/l and 555.10%, respectively, with assay specificities of 84.9 and 89.0%. The TBII assay predictive power (area under the curve (AUC)=0.817; 95% confidence interval (CI) =0.732-0.902) was equivalent to the TSI bioassay (AUC=0.868, 95% CI=0.803-0.934) (P=0.203). CONCLUSIONS: The predictive power of the third-generation TBII assay and Mc4-TSI bioassay are similarly strong. Measurement of TRAb using either third-generation TBII or Mc4-TSI in early GO periods would provide important prognostic information on future GO course.

Perpendicular magnetic anisotropy in amorphous ferromagnetic CoSiB/Pt multilayers.

Magnetic anisotropy properties of amorphous ferromagnetic CoSiB/Pt multilayers with perpendicular magnetic anisotropy (PMA, K(u)) were systematically investigated as a function of CoSiB layer thickness (t(coSiB)) and Pt layer thickness (t(Pt)). In two series of [CoSiB t(coSiB)Pt t(P1)]5 multilayers, the perpendicular coercivity (H(c)) increased to reach a maximum and then decreased with further increase in both t(coSiB) and t(Pt), due to intermixing of CoSiB/Pt interfaces. Particularly, using the amorphous soft magnetic CoSiB, the coercivity became very sensitive to the CoSiB thickness. These multilayer films exhibited a high K(u) of 2 x 10(6) erg/cc and a high H(c) of 360 Oe with marked squareness. It was found that even after annealing at 350 degrees C, the CoSiB/Pt multilayers had a high PMA and their H(c) increased.

The incidence, risk factors and prognostic implications of venous thromboembolism in patients with gastric cancer.

BACKGROUND: Data on venous thromboembolism (VTE) in gastric cancer (GC) are very scarce. OBJECTIVE: To investigate the incidence, risk factors and prognostic implications of VTE in Asian GC patients. METHODS: Prospective databases containing clinical information on GC patients (n = 2,085) were used. RESULTS: The 2-year cumulative incidences of all VTE events were 0.5%, 3.5% and 24.4% in stages I, II-IV(M0) and IV(M1), respectively. Advanced stage, older age and no major surgery were independent risk factors for developing VTE. When the VTE cases were classified into extremity venous thrombosis (EVT), pulmonary thromboembolism (PTE) or intra-abdominal venous thrombosis (IVT), IVTs (62%) were more common than EVTs (21%) or PTEs (17%). Although peri-operative pharmacologic thromboprophylaxis was not routinely administered, the VTE incidence after major surgery was only 0.2%. During chemotherapy, EVT/PTE developed more frequently than IVT (54% vs. 19%); however, during untreated or treatment-refractory periods, IVT developed more frequently than EVT/PTE (69% vs. 36%). In multivariate models, the development of EVT/PTE was a significant predictor of early death when compared with no occurrence of VTE (P < 0.05). However, IVT did not affect survival. CONCLUSION: This is the largest study that specially focused on VTE in GC and the VTE incidence in Asian GC patients was first demonstrated. Considering the low incidence of post-operative VTE development, the necessity of peri-operative pharmacologic thromboprophylaxis should be evaluated separately in Asian patients. The clinical situation of the development of EVT/PTE and IVT differed. Only EVT/PTE had an adverse effect on survival and IVT had no prognostic significance.

Deregulation of Cdk2 causes Bim-mediated apoptosis in p53-deficient tumors following actin damage.

We previously reported that actin damage by treatment with an actin-depolymerizing agent including pectenotoxin-2 induces Bim-mediated apoptosis in p53-deficient human tumors. In this study, we investigated a molecular mechanism underlying Bim-mediated apoptosis of p53-deficient tumor cells following actin damage. We found that actin inhibitors increased the protein levels of p53 and p21 and thereby inactivated both Cdk2 and Cdc2 kinases. However, p53- or p21-knockout cells fail to induce p21 and hence kept both Cdk2 and Cdc2 kinases active even after treatment with actin inhibitor. The p53- or p21-knockout cells became multinucleate and polyploidy in association with induction of apoptosis. Expression of Bcl-x(L) resulted in accumulation of polyploid cells in association with inhibition of apoptosis. However, expression of a dominant negative mutant (Cdk2dn) and treatment with chemical inhibitors for Cdk2 suppressed not only accumulation of multinucleated cells, but also induction of Bim expression and apoptosis. Therefore, these results suggest that Bim-mediated apoptosis following actin damage due to deregulation of Cdk2 and the cell cycle by the absence of functional p53.

Stable multidomain structures formed in the process of magnetization reversal in GaMnAs ferromagnetic semiconductor thin films.

The process of magnetization reversal in ferromagnetic Ga(1-x)Mn(x)As epilayers has been systematically investigated using the planar Hall effect (PHE). Interestingly, we have observed a pronounced asymmetry in the PHE hysteresis when the range of the field scan is restricted to fields below the final magnetization transition. The observed behavior indicates that (a) multidomain structures are formed as M undergoes a reorientation, (b) the domain landscape formed in this way remains stable even after the magnetic field is switched off, and (c) the reorientation of magnetization directions corresponding to the transition points in PHE takes place separately within each domain.

p53 and its homologues, p63 and p73, induce a replicative senescence through inactivation of NF-Y transcription factor.

Recent studies have identified two p53 homologues, p63 and p73. They activate p53-responsive promoters and induce apoptosis when overexpressed in certain human tumors. Here, we report that p63, like p53 and p73, induces replicative senescence when expressed in a tetracycline-regulated manner in EJ cells lacking a functional p53. In addition to transcription activation of p53-responsive genes, we found that p63 and p73 repress transcription of the cdk1 and cyclin B genes, both of which are irreversibly repressed in senescent human fibroblast. In transient transfection assay, p63 and p73 repress the cdk1 promoter regardless of the presence of a dominant negative mutant form of p53. Furthermore, we found that DNA binding activity of NF-Y transcription factor, which is essential for transcription of the cdk1 and cyclin B genes and inactivated in senescent fibroblast, is significantly decreased by expression of either of p53, p63, or p73. Since NF-Y binds to many promoters besides the cdk1 and cyclin B promoters, inactivation of NF-Y by p53 family genes may be a general mechanism for transcription repression in replicative senescence.

Mutational and kinetic evaluation of conserved His-123 in dual specificity protein-tyrosine phosphatase vaccinia H1-related phosphatase: participation of Tyr-78 and Thr-73 residues in tuning the orientation of His-123.

Active-site cysteine strategically positioned in the P-loop of protein-tyrosine phosphatases has been suggested to be further stabilized by hydrogen bonding arrays radiating out from the P-loop to neighboring residues. In this work, we investigated the structural role of histidine array in HC(X)(5)RS motif of the vaccinia H1-related protein phosphatase (VHR), using site-directed mutagenesis in conjunction with an extensive kinetic analysis. Conserved His-123 was mutated along with neighboring residues Tyr-78 and Thr-73. The increased pK(a) values of active-site Cys-124 found in Y78F and T73A mutants (6.51 and 6.75, respectively) were comparable to those of H123A and H123F mutants. Kinetic evaluation of Y78F and T73A mutants further implicates that the mutations perturb the relative position of Cys-124 within the P-loop. These results imply that Tyr-78 and Thr-73 make up an essential part of the His-123 array and structurally tune the Cys-124 position. Tyr-78 of VHR turns out to be the invariant Tyr reported in several protein-tyrosine phosphatases by a structure-based sequence alignment. Therefore, orientation of the imidazole ring of His-123 by the invariant Tyr-78 is crucial for maintaining the proper position of Cys-124 in the P-loop.

Pimarane cyclooxygenase 2 (COX-2) inhibitor and its structure-activity relationship.

The structure-activity relationship and molecular modelings of a novel pimarane COX-2 inhibitor are reported. Particularly, a series of linker extended analogues designed on the basis of these studies exhibited significantly enhanced COX-2 inhibitory activities and selectivities.

Isolation of a potent anti-MRSA sesquiterpenoid quinone from Ulmus davidiana var. japonica.

A highly potent anti-MRSA sesquiterpenoid has been isolated from Ulmus davidiana var. japonica, which has been traditionally used to treat infectious diseases in Korea. This naturally occurring antibiotic was identified as mansonone F (1). This compound has been found to be highly active specifically against MRSA and showed an MIC range of 0.39-3.13 microg/ml which is comparable to that of vancomycin.

The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair.

BACKGROUND: RNA of RNA-DNA hybrids can be degraded by ribonucleases H present in all organisms including the eukaryote Saccharomyces cerevisiae. Determination of the number and roles of the RNases H in eukaryotes is quite feasible in S. cerevisiae. RESULTS: Two S. cerevisiae RNases H, related to Escherichia coli RNase HI and HII, are not required for growth under normal conditions, yet, compared with wild-type cells, a double-deletion strain has an increased sensitivity to hydroxyurea (HU) and is hypersensitive to caffeine and ethyl methanesulphonate (EMS). In the absence of RNase H1, RNase H2 activity increases, and cells are sensitive to EMS but not HU and are more tolerant of caffeine; the latter requires RNase H2 activity. Cells missing only RNase H2 exhibit increased sensitive to HU and EMS but not caffeine CONCLUSIONS: Mutant phenotypes infer that some RNA-DNA hybrids are recognized by both RNases H1 and H2, while other hybrids appear to be recognized only by RNase H2. Undegraded RNA-DNA hybrids have an effect when DNA synthesis is impaired, DNA damage occurs or the cell cycle is perturbed by exposure to caffeine suggesting a role in DNA replication/repair that can be either beneficial or detrimental to cell viability.

A micromachined silicon depth probe for multichannel neural recording.

A process of making a new type of silicon depth-probe microelectrode array is described using a combination of plasma and wet etch. The plasma etch, which is done using a low temperature oxide (LTO) mask, enables probe thickness to be controlled over a range from 5 to 90 mu. Bending tests show that the probe's mechanical strength depends largely on shank thickness. More force can be applied to thicker shanks while thinner shanks are more flexible. One can then choose a thickness and corresponding mechanical strength using the process developed. The entire probe shaping process is performed only at low temperature, and thus is consistent with the standard CMOS fabrication. Using the probe in recording from rat's somatosensory cortex, we obtained four channel simultaneous recordings which showed clear independence among channels with a signal-to-noise ratio performance comparable with that obtained using other devices.

Constitutive activation of cyclin B1-associated cdc2 kinase overrides p53-mediated G2-M arrest.

Recent studies have suggested that p53 regulates the G2 checkpoint in the cell cycle and that this function is required for the maintenance of genomic integrity. In this study, we investigated a regulatory role of p53 specifically in G2-M transition. Human bladder carcinoma cells lacking functional p53 were synchronized at G1-S, which is preceded by p53-mediated G1 arrest. p53 expression in the synchronized cells was induced by infection with a recombinant adenovirus that encodes p53. After release from the G1-S arrest, the cells progressed to S-phase and G2 but failed to enter mitosis. Biochemical analysis showed that p53 inhibits cell cycle-dependent expression of cdc2 and cyclin B1 and consequently inhibits cdc2 kinase. The role of cyclin B1-associated cdc2 kinase in p53-mediated G2-M arrest was further investigated by expression of both cyclin B1 and cdc2AF, in which inhibitory phosphorylation sites were substituted. The cells expressing both cdc2AF and cyclin B1 showed a constitutive activation of cdc2 kinase during cell cycle progression and passed through G2-M regardless of p53 expression. Therefore, inactivation of cdc2 kinase through cdc2 and cyclin B1 repression is an essential step in p53-mediated G2-M arrest.

p53 negatively regulates cdc2 transcription via the CCAAT-binding NF-Y transcription factor.

The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We have reported previously that inducible expression of the p53 gene leads to the cell cycle arrest both at G(1) and G(2)/M in association with induction of p21 and reduction of mitotic cyclins (cyclin A and B) and cdc2 mRNA. In this study, we investigated the mechanism by which p53 regulates transcription of the cdc2 gene. Transient transfection analysis showed that wild type p53 represses whereas various dominant negative mutants of p53 increase cdc2 transcription. The cdc2 promoter activity is not repressed in cells transfected with a transactivation mutant, p53(22/23). An adenovirus oncoprotein, E1B-55K inhibits the p53-mediated repression of the cdc2 promoter, while E1B-19K does not. Since the cdc2 promoter does not contain a TATA sequence, we performed deletion and point mutation analyses and identified the inverted CCAAT sequence located at -76 as a cis-acting element for the p53-mediated regulation. We found that a specific DNA-protein complex is formed at the CCAAT sequence and that this complex contains the NF-Y transcription factor. Consistently, a dominant negative mutant of the NF-YA subunit, NF-YAm29, decreases the cdc2 promoter, and p53 does not further decrease the promoter activity in the presence of NF-YAm29. These results suggest that p53 negatively regulates cdc2 transcription and that the NF-Y transcription factor is required for the p53-mediated regulation.

Total synthesis of sufentanil.

Sufentanil, a potent anilidopiperidine analgesic, was synthesized from a simple thiophenylethylamine via six step sequence. The key parts of this synthesis involved an efficient construction of thiophenylethylpiperidone by aminomethano desilylation-cyclization followed by Swern oxidation and a direct regioselective N-nucleophilic spiral epoxide cleavage with aniline promoted by Lewis acids.

Adenovirus-mediated p53 tumor suppressor gene therapy against subcutaneous HuH7 hepatoma cell line nodule of nude mice.

Mutations of the tumor-suppressor gene p53 have been found in 30-50% cases of hepatocellular carcinoma (HCC). In this study, E1-negative adenoviral vector encoding wild-type p53 under the control of the human cytomegalovirus promoter (AdCMV-p53w) was constructed to evaluate its therapeutic efficacy against tumor nodules developing after injection of HuH7 cell lines in ten nude mice. When each nodule had reached 10 mm in perpendicular diameter, 1.5 x 10(8) pfu of AdCMV-p53w per session was injected intratumorally as follows: In group I (n=3), five sessions were injected every other day. In group II (n=3), only one session. Group III (n=4) as negative controls. The mice were sacrificed at 28 days post AdCMV-p53w injection. Tumor growth was significantly suppressed and delayed in group I and II compared to group III as compared by tumor volume at the end of observation. These results suggest that AdCMV-p53w may not only be effective in treating HCCs expressing mutant p53, but also useful as a local injectable gene therapy.