Studies on Nucleosides: Part XXVIII1. Synthesis of 4-Amino (or Hydroxy)-6-Methylthio-1-(3′-Deoxy-β- D-ribofuranosyl)-1-H-pyrazolo[3, 4-d]Pyrimidines

Abstract

4-Amino-6-methylthio-1-(3′-deoxy-β-D-ribofuranosyl)-1H-pyrazolo-[3, 4-d]pyrimidine (11) and 6-methylthio-4(5H)-oxo-1-(3′-deoxy-β-D-ribofuranosyl)-1H-pyrazolo[3, 4-d]pyrimidine (12) have been synthesized from 1, 2-di-O-acetyl-5-O-benzoyl-3-deoxyribofuranose (5) and 4, 6-bis (methylthio)-1H-pyrazolo-[3, 4-d]pyrimidine (6). in a convergent fashion. Structural proofs are based on MS, IR, ¹H NMR, ¹³C NMR and elemental analyses.     

Influence of ABCB1 genetic variants in breast cancer treatment outcomes

BackgroundTransportation of anticancer drugs such as anthracyclines across the membrane is regulated by P-glycoprotein encoded by the human multidrug resistance gene 1 (ABCB1). Polymorphisms in the ABCB1 gene (1236C > T, 2677G > T/A, 3435C > T) have been found to be associated with intrinsic and acquired cross resistance to these anticancer drugs. Therefore, the aim of this study is to evaluate the influence of ABCB1 gene polymorphisms in breast cancer treatment outcomes in terms of response and toxicity.MethodResponse to neo-adjuvant chemotherapy was evaluated in 100 patients while grade 2–4 toxicity was followed in 207 patients, who had undergone FEC/FAC chemotherapy. Genotyping for ABCB1 polymorphisms was done by PCR-RFLP. Chi square and logistic regression analyses were used to calculate Odd's ratio using SPSS ver 17.0. A meta analysis was also performed using Comprehensive Meta Analysis Ver 2.ResultsIn response evaluation, 1236C > T polymorphism was significantly associated with treatment response for CT genotype [OR = 5.17(1.3–20.2), P = 0.018] and in dominant model (CC vs CT + TT) [OR = 4.63(1.25–17.0), P = 0.021]. In the toxicity group, the T allele of 1236C>T was associated with grade 2–4 tocxicity [OR 1.48(1.00–2.20), P = 0.049] and the association was also significant in the recessive model [OR 1.88(1.05–3.39), P = 0.033]. For other two SNPs 2677G>T/A and 3435C>T no association was seen with either treatment response or grade 2–4 toxicity. In meta analysis, no overall association was found.ConclusionIn our study, significant association was seen for ABCB1 1236C>T polymorphism with treatment response. The meta analysis did not show overall association with treatment outcomes.     

In Vivo Imaging with Fluorescent Smart Probes to Assess Treatment Strategies for Acute Pancreatitis

Background and Aims

Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis.

Methods

Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent “smart” probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L-lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging.

Results

We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence-based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio.

Conclusions

We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition.     

Diagnostic Performance of Various Tests and Criteria Employed in Allergic Bronchopulmonary Aspergillosis: A Latent Class Analysis

Aim

The efficiency of various investigations and diagnostic criteria used in diagnosis of allergic bronchopulmonary aspergillosis (ABPA) remain unknown, primarily because of the lack of a gold standard. Latent class analysis (LCA) can provide estimates of sensitivity and specificity in absence of gold standard. Herein, we report the performance of various investigations and criteria employed in diagnosis of ABPA.

Methods

Consecutive subjects with asthma underwent all the following investigations Aspergillus skin test, IgE levels (total and A.fumigatus specific), Aspergillus precipitins, eosinophil count, chest radiograph, and high-resolution computed tomography (HRCT) of the chest. We used LCA to estimate the performance of various diagnostic tests and criteria in identification of ABPA.

Results

There were 372 asthmatics with a mean age of 35.9 years. The prevalence of Aspergillus sensitization was 53.2%. The sensitivity and specificity of various tests were Aspergillus skin test positivity (94.7%, 79.7%); IgE levels>1000 IU/mL (97.1%, 37.7%); A.fumigatus specific IgE levels>0.35 kUA/L (100%, 69.3%); Aspergillus precipitins (42.7%, 97.1%); eosinophil count>1000 cells/µL (29.5%, 93.1%); chest radiographic opacities (36.1%, 92.5%); bronchiectasis (91.9%, 80.9%); and, high-attenuation mucus (39.7%, 100%). The most accurate criteria was the Patterson criteria using six components followed by the Agarwal criteria. However, there was substantial decline in accuracy of the Patterson criteria if components of the criteria were either increased or decreased from six.

Conclusions

A.fumigatus specific IgE levels and high-attenuation mucus were found to be the most sensitive and specific test respectively in diagnosis of ABPA. The Patterson criteria remain the best diagnostic criteria however they have good veridicality only if six criteria are used.     

Catalysis by N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase (PIG-L) from Entamoeba histolytica: NEW ROLES FOR CONSERVED RESIDUES*

We showed previously that Entamoeba histolytica PIG-L exhibits a novel metal-independent albeit metal-stimulated activity. Using mutational and biochemical analysis, here we identify Asp-46 and His-140 of the enzyme as being important for catalysis. We show that these mutations neither affect the global conformational of the enzyme nor alter its metal binding affinity. The defect in catalysis, due to the mutations, is specifically due to an effect on V_max and not due to altered substrate affinity (or K_m). We propose a general acid-base pair mechanism to explain our results.     

QTL mapping in three tropical maize populations reveals a set of constitutive and adaptive genomic regions for drought tolerance

Despite numerous published reports of quantitative trait loci (QTL) for drought-related traits, practical applications of such QTL in maize improvement are scarce. Identifying QTL of sizeable effects that express more or less uniformly in diverse genetic backgrounds across contrasting water regimes could significantly complement conventional breeding efforts to improve drought tolerance. We evaluated three tropical bi-parental populations under water-stress (WS) and well-watered (WW) regimes in Mexico, Kenya and Zimbabwe to identify genomic regions responsible for grain yield (GY) and anthesis-silking interval (ASI) across multiple environments and diverse genetic backgrounds. Across the three populations, on average, drought stress reduced GY by more than 50 % and increased ASI by 3.2 days. We identified a total of 83 and 62 QTL through individual environment analyses for GY and ASI, respectively. In each population, most QTL consistently showed up in each water regime. Across the three populations, the phenotypic variance explained by various individual QTL ranged from 2.6 to 17.8 % for GY and 1.7 to 17.8 % for ASI under WS environments and from 5 to 19.5 % for GY under WW environments. Meta-QTL (mQTL) analysis across the three populations and multiple environments identified seven genomic regions for GY and one for ASI, of which six mQTL on chr.1, 4, 5 and 10 for GY were constitutively expressed across WS and WW environments. One mQTL on chr.7 for GY and one on chr.3 for ASI were found to be ‘adaptive’ to WS conditions. High throughput assays were developed for SNPs that delimit the physical intervals of these mQTL. At most of the QTL, almost equal number of favorable alleles was donated by either of the parents within each cross, thereby demonstrating the potential of drought tolerant × drought tolerant crosses to identify QTL under contrasting water regimes.     

Spectroscopic (FTIR,FT-Raman,NMR and UV) and molecular structure investigations of 1,5-diphenylpenta-2,4-dien-1-one: a combined experimental and theoretical approach

The title molecule 1,5-diphenylpenta-2,4-dien-1-one (cinnamylideneacetophenone, CA) has been synthesised and characterised by FTIR, FT-Raman, NMR and UV–vis spectral analyses. The possible stable conformers of the CA molecule were searched by potential energy surface scan at B3LYP level of theory. The molecular geometry from X-ray determination of the CA molecule in the ground state has been compared using the density functional theory (DFT) with 6-31G(d,p) basis set. The harmonic vibrational modes, the corresponding wavenumbers and IR and Raman intensities of most stable conformer were calculated by the DFT method. The assignments of the fundamentals were proposed on the basis of total energy distribution calculations. The calculated ¹³C and ¹H NMR chemical shifts using gauge including atomic orbitals approach are in good agreement with the observed chemical shifts. The molecular stability and bond strength have been investigated by applying natural bond orbital analysis. Using the time-dependent DFT method, the electronic absorption spectrum of the title compound has been predicted and the electronic transitions within the molecule have been interpreted. The molecular electrostatic potential map was used for predicting possible hydrogen and oxygen bonding sites in the CA molecule.     

Impact of buserelin acetate or hCG administration on day 12 post-ovulation on subsequent luteal profile and conception rate in buffalo (Bubalus bubalis)

The present study investigated the impact of gonadotropic hormone administration on day 12 post-ovulation on subsequent luteal profile and conception rate in buffaloes. All the buffaloes (n = 48) were estrus synchronized by a synthetic analogue of prostaglandin F_2α (PGF_2α), administered 11 days apart, followed by insemination during mid to late estrus. To examine the effect of mid-luteal phase hormonal treatment, buffaloes were randomly divided into control (normal saline, n = 14), d12-BA (buserelin acetate, 20 μg, n = 17) and d12-hCG (hCG, 3000 IU, n = 17) groups. Ovaries were scanned on the day of induced estrus to measure the preovulatory follicle (POF) diameter and on days 5, 12, 16 and 21 post-ovulation to examine the alterations in corpus luteum (CL) diameter. On the day of each sonography, blood samples were collected for the estimation of plasma progesterone. In treatment groups, luteal profile (CL diameter and plasma progesterone) on day 16–21 post-ovulation was better (P < 0.05) as well as first service conception rate was higher (52.9% in each treatment group vs. 28.6%, P > 0.05) compared to controls. All the pregnant buffaloes exhibited higher (P < 0.05) plasma progesterone on various post-ovulation days than their respective non-pregnant counterparts. Treatment-induced accessory corpus luteum (ACL) formation was observed in 58.8 per cent and 70.6 per cent buffaloes of d12-BA and d12-hCG group, respectively, that also had higher (P < 0.05) plasma progesterone compared to controls. Compared to the spontaneous CL, the diameter of ACL was less (P < 0.05) in the treatment groups. In conclusion, buserelin acetate and hCG administration on day 12 post-ovulation leads to accessory CL formation, improves luteal profile and consequently increases conception rate in buffaloes.     

The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling

Type III receptor tyrosine kinases (RTKs), FLT3 and c-Kit play important roles in a variety of cellular processes. A number of SH2-domain containing proteins interact with FLT3 and c-Kit and regulate downstream signaling. The SH2-domain containing non-receptor protein tyrosine kinase CSK is mainly studied in the context of regulating Src family kinases. Here we present an additional role of this kinase in RTK signaling. We show that CSK interacts with FLT3 and c-Kit in a phosphorylation dependent manner. This interaction is facilitated through the SH2-domain of CSK. Under basal conditions CSK is mainly localized throughout the cytosolic compartment but upon ligand stimulation it is recruited to the inner side of cell membrane. CSK association did not alter receptor ubiquitination or phosphorylation but disrupted downstream signaling. Selective depletion of CSK using siRNA, or inhibition with CSK inhibitor, led to increased phosphorylation of Akt and Erk, but not p38, upon FLT3 ligand (FL) stimulation. Stem cell factor (SCF)-mediated Akt and Erk activation was also elevated by CSK inhibition. However, siRNA mediated CSK knockdown increased SCF stimulated Akt phosphorylation but decreased Erk phosphorylation. CSK depletion also significantly increased both FL- and SCF-induced SHC, Gab2 and SHP2 phosphorylation. Furthermore, CSK depletion contributed to oncogenic FLT3- and c-Kit-mediated cell proliferation, but not to cell survival. Thus, the results indicate that CSK association with type III RTKs, FLT3 and c-Kit can have differential impact on receptor downstream signaling.     

Evaluation of steroidal amines as lipid raft modulators and potential anti-influenza agents

The influenza A virus (IFV) possesses a highly ordered cholesterol-rich lipid envelope. A specific composition and structure of this membrane raft envelope are essential for viral entry into cells and virus budding. Several steroidal amines were investigated for antiviral activity against IFV. Both, a positively charged amino function and the highly hydrophobic (C log P ? 5.9) ring system are required for IC₅₀ values in the low μM range. An amino substituent is preferential to an azacyclic A-ring. We showed that these compounds either disrupt or augment membrane rafts and in some cases inactivate the free virus. Some of the compounds also interfere with virus budding. The antiviral selectivity improved in the series 3-amino, 3-aminomethyl, 3-aminoethyl, or by introducing an OH function in the A-ring. Steroidal amines show a new mode of antiviral action in directly targeting the virus envelope and its biological functions.