A triterpenediol from <Emphasis Type="Italic">Boswellia serrata</Emphasis> induces apoptosis through both the intrinsic and extrinsic apoptotic pathways in human leukemia HL-60 cells

A triterpenediol (TPD) comprising of isomeric mixture of 3α, 24-dihydroxyurs-12-ene and 3α, 24-dihydroxyolean-12-ene from Boswellia serrata induces apoptosis in cancer cells. An attempt was made in this study to investigate the mechanism of cell death by TPD in human leukemia HL-60 cells. It inhibited cell proliferation with IC₅₀ ∼ 12 μg/ml and produced apoptosis as measured by various biological end points e.g. increased sub-G0 DNA fraction, DNA ladder formation, enhanced AnnexinV-FITC binding of the cells. Further, initial events involved massive reactive oxygen species (ROS) and nitric oxide (NO) formation, which were significantly inhibited by their respective inhibitors. Persistent high levels of NO and ROS caused Bcl-2 cleavage and translocation of Bax to mitochondria, which lead to loss of mitochondrial membrane potential (Δψ_m) and release of cytochrome c, AIF, Smac/DIABLO to the cytosol. These events were associated with decreased expression of survivin and ICAD with attendant activation of caspases leading to PARP cleavage. Furthermore, TPD up regulated the expression of cell death receptors DR4 and TNF-R1 level, leading to caspase-8 activation. These studies thus demonstrate that TPD produces oxidative stress in cancer cells that triggers self-demise by ROS and NO regulated activation of both the intrinsic and extrinsic signaling cascades.     

Structure–activity relationship (SAR) of parthenin analogues with pro-apoptotic activity: Development of novel anti-cancer leads

Analogues of parthenin were synthesized by substitutions at different reaction centres to establish a structure–activity relationship (SAR). Some of the molecules have displayed significant cytotoxicity in human cervical carcinoma (HeLa) and human myeloid leukemia (HL-60) cells. A few of the compounds also induced apoptosis in HL-60 cells measured in terms of sub-Go/G1 DNA fraction. Also one of the lead molecules has been shown to be the inhibitor of both telomerase and topoisomerase-II.     

An improved method for the hydrolysis of hardwood carbohydrates to monomers

The range of xylan content reported for sugar maple (Acer saccharum) is wider than for most hardwoods. Credible values have been reported that range from less than 16 wt% to more than 19 wt% based on extractive-free wood. Carbohydrate composition of biomass is normally determined by a two stage H₂SO₄ hydrolysis followed by quantification of the sugar monomers. Acetic and uronic acids are also quantified for xylan-rich materials. In this research, the H₂SO₄ hydrolysis conditions were modified and an average xylan content of 18.7% was obtained for sugar maple with a standard deviation (SD) of 0.41% for eight analyses. Minor refinements were made and an average of 18.6% (SD = 0.29%) was obtained for another eight analyses. On this occasion the acetyl to xylose mole ratio was 0.71 with a standard deviation of only 0.008. Proton NMR was utilized in quantifying sugar monomers and acetic acid. The modified hydrolysis procedure gave all the expected results for Betula papyrifera, a species without much controversy regarding its carbohydrate composition. A high percentage of glucan plus xylan was also obtained for an experimentally grown poplar with a low lignin content of 17.7%. The summative analyses varied from 99.7% to 101.5% for the three species.     

Semiautomated high-sensitivity profiling of human blood serum glycoproteins through lectin preconcentration and multidimensional chromatography/tandem mass spectrometry

We describe an effective analytical approach to identify trace glycoproteins in a small volume of human serum. The system is based on automatable affinity enrichment through silica-based lectin microcolumns and a further separation of the retained glycoproteins on a reversed-phase liquid chromatography with superficially porous packing, operating at high temperature. The fractionated sample is further directed into a 96-well plate for trypsinization and LC-MS/MS analysis. Using a major-component depleted blood serum (16 microg total protein), we were able to identify 271 glycoproteins through this analytical system.     

The HIV Care Continuum: Changes over Time in Retention in Care and Viral Suppression

Background

The HIV care continuum (diagnosis, linkage to care, retention in care, receipt of antiretroviral therapy (ART), viral suppression) has been used to identify opportunities for improving the delivery of HIV care. Continuum steps are typically calculated in a conditional manner, with the number of persons completing the prior step serving as the base population for the next step. This approach may underestimate the prevalence of viral suppression by excluding patients who are suppressed but do not meet standard definitions of retention in care. Understanding how retention in care and viral suppression interact and change over time may improve our ability to intervene on these steps in the continuum.

Methods

We followed 17,140 patients at 11 U.S. HIV clinics between 2010-2012. For each calendar year, patients were classified into one of five categories: (1) retained/suppressed, (2) retained/not-suppressed, (3) not-retained/suppressed, (4) not-retained/not-suppressed, and (5) lost to follow-up (for calendar years 2011 and 2012 only). Retained individuals were those completing ≥2 HIV medical visits separated by ≥90 days in the year. Persons not retained completed ≥1 HIV medical visit during the year, but did not meet the retention definition. Persons lost to follow-up had no HIV medical visits in the year. HIV viral suppression was defined as HIV-1 RNA ≤200 copies/mL at the last measure in the year. Multinomial logistic regression was used to determine the probability of patients’ transitioning between retention/suppression categories from 2010 to 2011 and 2010 to 2012, adjusting for age, sex, race/ethnicity, HIV risk factor, insurance status, CD4 count, and use of ART.

Results

Overall, 65.8% of patients were retained/suppressed, 17.4% retained/not-suppressed, 10.0% not-retained/suppressed, and 6.8% not-retained/not-suppressed in 2010. 59.5% of patients maintained the same status in 2011 (kappa=0.458) and 53.3% maintained the same status in 2012 (kappa=0.437).

Conclusions

Not counting patients not-retained/suppressed as virally suppressed, as is commonly done in the HIV care continuum, underestimated the proportion suppressed by 13%. Applying the care continuum in a longitudinal manner will enhance its utility.     

Genomic and protein structural maps of adaptive evolution of human influenza A virus to increased virulence in the mouse

Adaptive evolution is characterized by positive and parallel, or repeated selection of mutations. Mouse adaptation of influenza A virus (IAV) produces virulent mutants that demonstrate positive and parallel evolution of mutations in the hemagglutinin (HA) receptor and non-structural protein 1 (NS1) interferon antagonist genes. We now present a genomic analysis of all 11 genes of 39 mouse adapted IAV variants from 10 replicate adaptation experiments. Mutations were mapped on the primary and structural maps of each protein and specific mutations were validated with respect to virulence, replication, and RNA polymerase activity. Mouse adapted (MA) variants obtained after 12 or 20-21 serial infections acquired on average 5.8 and 7.9 nonsynonymous mutations per genome of 11 genes, respectively. Among a total of 115 nonsynonymous mutations, 51 demonstrated properties of natural selection including 27 parallel mutations. The greatest degree of parallel evolution occurred in the HA receptor and ribonucleocapsid components, polymerase subunits (PB1, PB2, PA) and NP. Mutations occurred in host nuclear trafficking factor binding sites as well as sites of virus-virus protein subunit interaction for NP, NS1, HA and NA proteins. Adaptive regions included cap binding and endonuclease domains in the PB2 and PA polymerase subunits. Four mutations in NS1 resulted in loss of binding to the host cleavage and polyadenylation specificity factor (CPSF30) suggesting that a reduction in inhibition of host gene expression was being selected. The most prevalent mutations in PB2 and NP were shown to increase virulence but differed in their ability to enhance replication and demonstrated epistatic effects. Several positively selected RNA polymerase mutations demonstrated increased virulence associated with >300% enhanced polymerase activity. Adaptive mutations that control host range and virulence were identified by their repeated selection to comprise a defined model for studying IAV evolution to increased virulence in the mouse.     

Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.     

Simple micro-method for the isolation of gangliosides by reversed-phase chromatography

A simple and convenient technique has been developed for the isolation of gangliosides from small amounts of tissues or cells. A ganglioside fraction obtained by chromatography of the total lipid extract of DEAE-Sephadex was subjected to alkaline hydrolysis and salts and other non-lipid contaminants were removed by reversed-phase chromatography on a C₁₈ Sep-Pak cartridge. The purified gangliosides were then obtained by chromatography on a small Iatrobeads or Unisil column. This procedure yields a quantitative recovery of gangliosides that are free of contaminants which interfere with thin-layer chromatographic analysis. The procedure was used for the quantitative isolation of gangliosides from human brain white matter and human erythrocytes.     

Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2

beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.