Synthesis and cytotoxicity of 1,6,8,9-substituted α-carboline derivatives

α-Carboline (pyrido[2,3-b]indole) was selected as the basic scaffold for development of antileukemic agents by structural modification. From the structure-activity study, it was found that sequential introduction of 6-acetyl and 9-substituted benzyl groups onto an α-Carboline scaffold resulted in 6-acetyl-9-(3,5-dimethoxybenzyl)-9H-pyrido[2,3-b]indole and 6-acetyl-9-(3,4,5-trimethoxybenzyl)-9H-pyrido[2,3-b]indole with potent cytotoxicity against the HL-60 cell line. These two compounds will be used as new lead compounds for further investigation.     

Synthesis of an enzyme—dependent prodrug and evaluation of its potential for colon targeting

AIM：To synthesize dexamethasone-succinate-dextran(DSD)conjugate and to evaluate the potentiality of DSD for the treatment of inflammatory bowel diseases.METHODS:Dexamethasone was attached to dextran(average molecular weight=70400Dalton)using succinate anhydride in an an hydrous environment catalyzed by 4-dimethylaminopyridine and1,1′-carbornyldiimidazole.The chemical structure of DSDwas identified by UV,IR and NMR,and the in vivo drug release behavior of this prodrug was investigated after oral administration of DNA suspension.RESULTS:The DSD conjugate was obtained in two steps and the content of dexamethasone in DSD was11.28%.The dextran prodrug was stable in rat stomach and smal intestine and negligibly absorbed form these tracts.Fout to nine hours after the oral administration.most of the prodrug(>95%)had moved to the cecum and colon,and was easily hydrolyzed by an endodextranase.Recover of dexamethasone from colon and cecum after administration of DSD conjugate was 6-12folds higher than the recovery after administration of unmodified dexamethasone(t=2.74,P<0.050.The prefernetial release of free dexamethasone in cecum and colon over that in the small intestine was staistically significant(t=2.27,P<0.05).CONCLUSION:The results of this study indicate that dependentconjugates may be useful in selectively delivering glucocorticoids to the colon.     

Synthesis and Properties of a Chiroptically Active Oligomer from 3,4-Ethylenedioxythiophene and (–)-Myrtenal

Oxidative polycondensation of 3,4-ethylenedioxythiophene and (–)-myrtenal was carried out with POCl₃. A π-conjugated system thus constructed consists of aromatic and quinoidal alternating structure linked via methine groups. We examined iodine doping effect for the resultant material with electron spin resonance spectroscopy. Circular dichroism spectra in chloroform solution showed blue-shift with increase of iodine concentration. This result indicates that the doping process can tune chiroptical activity of the chiral π-conjugated system.     

Mediastinal lymphadenectomy fulfilling NCCN criteria may improve the outcome of clinical N0–1 and pathological N2 non-small cell lung cancer

Background

This retrospective study investigated whether mediastinal lymphadenectomy compliant with the National Comprehensive Cancer Network (NCCN) criteria will improve the oncological outcomes of clinical early-stage lung cancer.

Methods

From 2003–2010, 712 consecutive cases of clinical N0/1 were included for retrospective analysis, including 152 confirmed cases of pN2 and 560 of pN0–1 disease following surgery. Group A was defined as the cases fulfilling NCCN lymphadenectomy criteria (≥ three stations of N2 nodes dissection) and group B included all other cases. The groups were stratified according to pN status and the outcomes were assessed.

Results

Five-year overall survival (OS) and 5-year disease-free survival (DFS) were significantly different between group A versus B [72%±2% vs. 63%±4% (OS), P=0.014; 58.0%±2% vs. 49%±4% (DFS), P=0.038] in the whole cohort. After stratification by pN status, this difference was remained in pN2 subgroup [50%±5% vs. 25%±9% (OS), P=0.006; 31.0%±4% vs. 13%±7% (DFS), P=0.014], but not in pN0–1 subgroups. Cox regression analysis showed that performing a lymphadenectomy fulfilling NCCN criteria was a significant prognostic factor for OS either in the whole cohort [P=0.003, hazard ratio (HR): 0.598, 95% confidence interval (CI): 0.425–0.841] or in patients of pN2 status (P=0.038, HR: 0.559, 95% CI: 0.323–0.968). Cases with ≥4 N2 stations dissected did not achieve better survival benefit compared to those harvesting 3 stations in cN0/1–pN2 group (P=0.152).

Conclusions

Mediastinal lymphadenectomy fulfilling NCCN criteria appears to improve the survival of unexpected N2 group (cN0/1-pN2) among early-stage lung cancer patients. More extended N2 node dissection may not further improve the outcome in this group.     

Flow cytometric evaluation of platelet–leukocyte conjugate stability over time: methodological implications of sample handling and processing

Journal of Thrombosis and Thrombolysis - Platelet activation and subsequent aggregation is a vital component of atherothrombosis resulting in acute myocardial infarction. Therefore, quantifying...     

Structural and mechanistic insights into the association of PKCα-C2 domain to PtdIns(4,5)P2

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca²⁺-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCα-C2 domain in complex with Ca²⁺, 1,2-dihexanoyl-sn-glycero-3-[phospho-l-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P₂] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P₂ occupies the concave surface of strands β3 and β4. Strikingly, the structure reveals a PtdIns(4,5)P₂-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns(4,5)P₂ severely impaired the ability of PKCα to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns(4,5)P₂ is presented.     

Phosphorylation of aquaporin-2 regulates its endocytosis and protein–protein interactions

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin–Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2, (iii) AQP2 protein–protein interactions, and (iv) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (t_1/2 = 5.1 h and t_1/2 = 4.4 h, respectively) compared to WT-AQP2 (t_1/2 = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein–protein interactions.     

Synthesis of granulocyte–macrophage colony-stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material

Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a medicinally important glycoprotein, used as an immunostimulant following bone-marrow transplant. On the basis of reports of its potential utility as an anticancer vaccine adjuvant, we undertook to develop a synthetic route toward single-glycoform GM-CSF. We describe herein a convergent total synthesis of GM-CSF aglycone and two homogeneous glycoforms. Analytical and biological studies confirm the structure and activity of these synthetic congeners.Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a secreted glycoprotein that promotes cellular growth and proliferation. GM-CSF signaling initiates a cascade that culminates in the production of white blood cells through stem-cell stimulation in the bone marrow (Fig. 1) (1). Therapeutically, this glycoprotein is valued for its properties as an immunostimulant; GM-CSF impacts the production, differentiation, and function of dendritic cells through potentiation of the CD4⁺ T-cell response (2, 3) and is regularly administered to patients undergoing chemotherapy or autologous bone-marrow transplant. GM-CSF is approved in Europe as the aglycone (Molgramostim) (4) and in the United States as a glycosylated, mutant form (Sargramostim). At present, glycosylated GM-CSF is obtained exclusively via recombinant technologies using yeast (Sargramostim) or Chinese hamster ovary (CHO) cell (Regramostim) technologies, which yield complex mixtures of glycoforms. The glycan heterogeneity reflects a lack of specificity in CHO-cell posttranslational glycosylation. The degree of GM-CSF glycosylation has been reported to affect the in vivo properties of the glycoprotein (5, 6); the aglycone may cause increased adverse side effects, perhaps due to its enhanced susceptibility to truncation pathways (7).Open in a separate windowFig. 1.GM-CSF 3D structure and fully glycosylated GM-CSF sequence (1). The ribbon structure of GM-CSF is based on the X-ray of GM-CSF aglycone expressed by Escherichia coli.In light of our longstanding interest in synthetic anticancer and HIV vaccines (8), we took note of reports suggesting that GM-CSF might serve as a useful vaccine immunoadjuvant. Administration of GM-CSF results in robust potentiation of the immune response, and clinical studies suggest that the glycoprotein may hold promise as an adjuvant for anticancer vaccines (9, 10). However, studies to date have used recombinantly derived GM-CSF mixtures, and results have been inconclusive (9); perhaps such translational issues might be addressed in a more informative fashion with homogeneous GM-CSF agents. In a more speculative line of inquiry, we also wonder whether appendage of tumor-associated carbohydrate antigens to the protein backbone might yield powerful new anticancer vaccine candidates (11). Even beyond these fascinating medicinal questions, we recognized in GM-CSF a synthetically compelling glycoprotein target. With these considerations in mind, we undertook to design a modular route to homogeneous GM-CSF glycoforms. In this endeavor, we would rely upon key methodological and strategic advances from many groups, including ours, in the synthesis of complex glycoproteins. The past decade has indeed witnessed a dramatic maturation of the field of “biologics” through chemical synthesis. Our recent synthesis of homogeneous erythropoietin, bearing complex glycan domains at each native position, is illustrative of the growing power of chemical synthesis to facilitate the study of medicinally relevant biologic targets (12–14). We describe herein the synthesis of homogeneous GM-CSF glycoproteins through a convergent route that offers ready access to a menu of glycoforms for further study.Structurally, GM-CSF consists of 127 aa with multiple sites of glycosylation (Fig. 1). Two N-linked glycans are located at Asn²⁷ and Asn³⁷ (15, 16). Interestingly, neither the position nor the extent of O-linked glycosylation has been unambiguously established. Studies suggest a range from two (Ser⁷ and Ser⁹/Thr¹⁰) to four (Ser⁵, Ser⁷, Ser⁹, and Thr¹⁰) sites of glycosylation (17). The tertiary structure is strongly influenced by two cross-linked disulfide bonds.     

The CHADS2 and CHA2DS2–VASc scores predict new occurrence of atrial fibrillation and ischemic stroke

Background

Early identification of individuals who are at risk of developing atrial fibrillation (AF) and ischemic stroke may enable a closer surveillance and thus prompt initiation of oral anticoagulation for stroke prevention.

Objective

This study sought to investigate whether congestive heart failure, hypertension, age?≥?75 years, diabetes, previous stroke (CHADS₂) and CHA₂DS₂–vascular disease, age 65–74 years, sex category (CHA₂DS₂–VASc) scores can predict new-onset AF and/or ischemic stroke in patients presenting with arrhythmic symptoms.

Methods and results

We prospectively followed up 528 patients (68.5?±?10.6 years, male 46.2 %) presented for assessment of arrhythmic symptoms but without any documented arrhythmia, including AF for development of new-onset AF and/or ischemic stroke. Their mean CHADS₂ and CHA₂DS₂–VASc scores on presentation were 1.3?±?1.3 and 2.3?±?1.5, respectively. After 6.1 years, 89 patients (16.8 %, 2.77 per 100 patient-years) had documented AF, and 65 patients (12.3 %, 2.0 per 100 patient-years) suffered stroke. Both the CHADS₂ (C statistic 0.63, 95 % confidence interval (CI) 0.58–0.67, P?2DS₂–VASc (C statistic 0.63, 95 % CI 0.59–0.67, P?2 (C statistic 0.69, 95 % CI 0.65–0.73, P?2DS₂–VASc (C statistic 0.69, 95 % CI 0.65–0.73, P?Conclusion The CHADS₂ and CHA₂DS₂–VASc scores can be used in patients who presented with arrhythmic symptoms to identify those who are at risk with developing new-onset clinical AF and ischemic stroke for close clinical surveillance and early intervention.     

The Role of C2–C7 and O–C2 Angle in the Development of Dysphagia After Cervical Spine Surgery

Dysphagia is a known complication of cervical surgery and may be prolonged or occasionally serious. A previous study showed that dysphagia after occipitocervical fusion was caused by oropharyngeal stenosis resulting from O–C2 (upper cervical lordosis) fixation in a flexed position. However, there have been few reports analyzing the association between the C2–C7 angle (middle-lower cervical lordosis) and postoperative dysphagia. The aim of this study was to analyze the relationship between cervical lordosis and the development of dysphagia after anterior and posterior cervical spine surgery (AC and PC). Three hundred fifty-four patients were reviewed in this retrospective clinical study, including 172 patients who underwent the AC procedure and 182 patients who had the PC procedure between June 2007 and May 2010. The presence and duration of postoperative dysphagia were recorded via face-to-face questioning or telephone interview performed at least 1 year after the procedure. Plain cervical radiographs before and after surgery were collected. The O–C2 angle and the C2–C7 angle were measured. Changes in the O–C2 angle and the C2-C7 angle were defined as dO–C2 angle = postoperative O–C2 angle ? preoperative O–C2 angle and dC2–C7 angle = postoperative C2–C7 angle ? preoperative C2–C7 angle. The association between postoperative dysphagia with dO–C2 angle and dC2–C7 angle was studied. Results showed that 12.8 % of AC and 9.4 % of PC patients reported dysphagia after cervical surgery. The dC2–C7 angle has considerable impact on postoperative dysphagia. When the dC2–C7 angle is greater than 5°, the chance of developing postoperative dysphagia is significantly greater. The dO–C2 angle, age, gender, BMI, operative time, blood loss, procedure type, revision surgery, most cephalic operative level, and number of operative levels did not significantly influence the incidence of postoperative dysphagia. No relationship was found between the dC2–C7 angle and the degree of dysphagia. We conclude that postoperative dysphagia is common after cervical surgery. The dC2–C7 angle may play an important role in the development of dysphagia in both anterior and posterior cervical spine surgery. Intraoperative measurement of the dC2–C7 angle is practical and essential in avoiding inadvertent postoperative dysphagia.     

The evaluation of the effectiveness of intra-articular steroid,tenoxicam, and combined steroid–tenoxicam injections in the treatment of patients with knee osteoarthritis

Clinical Rheumatology - Although intra-articular corticosteroid injections are widely applied in the treatment of knee osteoarthritis (OA), its effect is short term. Additionally, apart from oral...     

Meat and fish intake and type 2 diabetes: Dose–response meta-analysis of prospective cohort studies

AimsThis meta-analysis aimed to quantitatively examine the possible associations between total meat, red meat, processed meat, poultry and fish intakes and type 2 diabetes (T2D).MethodsRelevant articles were identified in PubMed, Embase and Web of Science databases using a search time up to January 2019. Generalized least-squares trend estimations and restricted cubic spline regression models were used for analysis.ResultsTwenty-eight articles were included in the analysis. When comparing the highest with the lowest category of meat intake, the summary relative risk of T2D was 1.33 (95% CI: 1.16–1.52) for total meat, 1.22 (95% CI: 1.16–1.28) for red meat, 1.25 (95% CI: 1.13–1.37) for processed meat, 1.00 (95% CI: 0.93–1.07) for poultry and 1.01 (95% CI: 0.93–1.10) for fish. In the dose–response analysis, each additional 100 g/day of total and red meat, and 50 g/day of processed meat, were found to be associated with a 36% (95% CI: 1.23–1.49), 31% (95% CI: 1.19–1.45) and 46% (95% CI: 1.26–1.69) increased risk of T2D, respectively. In addition, there was evidence of a non-linear dose–response association between processed meat and T2D (P = 0.004), with the risk increasing by 30% with increasing intakes up to 30 g/day.ConclusionOur meta-analysis has shown a linear dose–response relationship between total meat, red meat and processed meat intakes and T2D risk. In addition, a non-linear relationship of intake of processed meat with risk of T2D was detected.     

Prospective evaluation of insulin and incretin dynamics in obese adults with and without diabetes for 2 years after Roux-en-Y gastric bypass

Aims/hypothesis

In this prospective case–control study we tested the hypothesis that, while long-term improvements in insulin sensitivity (S_I) accompanying weight loss after Roux-en-Y gastric bypass (RYGB) would be similar in obese individuals with and without type 2 diabetes mellitus, stimulated-islet-cell insulin responses would differ, increasing (recovering) in those with diabetes but decreasing in those without. We investigated whether these changes would occur in conjunction with favourable alterations in meal-related gut hormone secretion and insulin processing.

Methods

Forty participants with type 2 diabetes and 22 participants without diabetes from the Longitudinal Assessment of Bariatric Surgery (LABS-2) study were enrolled in a separate, longitudinal cohort (LABS-3 Diabetes) to examine the mechanisms of postsurgical diabetes improvement. Study procedures included measures of S_I, islet secretory response and gastrointestinal hormone secretion after both intravenous glucose (frequently-sampled IVGTT [FSIVGTT]) and a mixed meal (MM) prior to and up to 24 months after RYGB.

Results

Postoperatively, weight loss and S_I-_FSIVGTT improvement was similar in both groups, whereas the acute insulin response to glucose (AIRglu) decreased in the non-diabetic participants and increased in the participants with type 2 diabetes. The resulting disposition indices (DI_FSIVGTT) increased by three- to ninefold in both groups. In contrast, during the MM, total insulin responsiveness did not significantly change in either group despite durable increases of up to eightfold in postprandial glucagon-like peptide 1 levels, and S_I-MM and DI_MM increased only in the diabetes group. Peak postprandial glucagon levels increased in both groups.

Conclusions/interpretation

For up to 2 years following RYGB, obese participants without diabetes showed improvements in DI that approach population norms. Those with type 2 diabetes recovered islet-cell insulin secretion response yet continued to manifest abnormal insulin processing, with DI values that remained well below population norms. These data suggest that, rather than waiting for lifestyle or medical failure, RYGB is ideally considered before, or as soon as possible after, onset of type 2 diabetes.

Trial registration

ClinicalTrials.gov NCT00433810

     

Flux-Aided Synthesis of Lu2O3 and Lu2O3:Eu—Single Crystal Structure,Morphology Control and Radioluminescence Efficiency

Li₂SO₄ or (Li₂SO₄ + SiO₂)-mixture fluxes were used to prepare a Lu₂O₃:Eu powder phosphor as well as an undoped Lu₂O₃ utilizing commercial lutetia and europia as starting reagents. SEM images showed that the fabricated powders were non-agglomerated and the particles sizes varied from single microns to tens of micrometers depending largely on the flux composition rather than the oxide(s)-to-flux ratio. In the presence of SiO₂ in the flux, certain grains grew up to 300–400 μm. The lack of agglomeration and the large sizes of crystallites allowed making single crystal structural measurements and analysis on an undoped Lu₂O₃ obtained by means of the flux technique. The cubic structure with a = 10.393(2) Å, and Ia3 space group at 298 K was determined. The most efficient radioluminescence of Lu₂O₃:Eu powders reached 95%–105% of the commercial Gd₂O₂S:Eu.     

Increased expression and function of the myocardial Na–K–2Cl cotransporter in failing rat hearts

Abstract Recent studies indicate a role of the Na–K–2Cl cotransporter (NKCC) in regulation of myocardial function. However, potential pathophysiological properties of NKCC in conditions like myocardial infarction (MI) and heart failure have not been explored. We investigated the cellular localization of myocardial NKCC and whether myocardial NKCC levels are changed upon induction of post-infarction heart failure in rats. Immunohistochemical analysis demonstrated extensive distribution of NKCC in normal rat myocardium with fairly strong expression in cardiomyocytes, fibroblasts, vascular endothelial cells, as well as smooth muscle cells. Myocardial mRNA levels of NKCC were investigated at 2, 7 and 28 days after induction of MI or sham operation, but no changes were found. Cardiomyocytes and non-cardiomyocytes were isolated 7 days after induction of MI or sham operation. An ∼2-fold increase of the NKCC mRNA levels was found in isolated cardiomyocytes from heart failure rats compared to that of sham-operated rats (P < 0.001), whereas a trend towards decreased mRNA levels of NKCC in isolated non-cardiomyocytes was observed. In addition, we found a bumetanide sensitive ⁸⁶Rb⁺ influx mechanism present in the hearts after induction of MI (P < 0.05). Thus, our data indicate cardiomyocyte specific increase in NKCC mRNA levels and increased NKCC activity in post-infarction heart failure. Our results may indicate a potential role of NKCC during post-infarction remodeling.