Differences in variation of human immunodeficiency virus type 1 sequences from Henan and Shanghai regions of China

Illegally paid blood donation was a risk factor for HIV acquisition exclusively in Henan and Hubei Provinces of China, and not in Shanghai. Nucleotide sequences in the gag and env genes of HIV-1 were compared between isolates from Henan and Shanghai regions of China to test whether an expected higher degree of a common source of infections from this unique blood donation transmission risk would be evident as decreased variation among Henan isolates in an exploratory cross-sectional analysis. Among 38 isolates studied, 23 of 23 (100%) from Henan and 8 of 15 (54%) from Shanghai were subtype B. In addition, fewer sequence differences were found in gp41 of subtype B isolates from Henan than from Shanghai isolates. Further studies with additional controls are therefore warranted to confirm the role of the degree of a common source of infections in differences in HIV variation across populations. Fundation items: The Vanderbilt-Meharry Center for AIDS Research (P30 AI 54999); R.T.D (R01 AI 29193); Start Fund of Ministry of Education of China (for Hong-zhou LU, 2004BA719A10).     

Stem cell aging is controlled both intrinsically and extrinsically in the Drosophila ovary

It is widely postulated that tissue aging could be, at least partially, caused by reduction of stem cell number, activity, or both. However, the mechanisms of controlling stem cell aging remain largely a mystery. Here, we use Drosophila ovarian germline stem cells (GSCs) as a model to demonstrate that age-dependent decline in the functions of stem cells and their niche contributes to overall stem cell aging. BMP signaling activity from the niche significantly decreases with age, and increasing BMP signaling can prolong GSC life span and promote their proliferation. In addition, the age-dependent E-cadherin decline in the stem cell-niche junction also contributes to stem cell aging. Finally, overexpression of SOD, an enzyme that helps eliminate free oxygen species, in either GSCs or their niche alone can prolong GSC life span and increase GSC proliferation. Therefore, this study demonstrates that stem cell aging is controlled extrinsically and intrinsically in the Drosophila ovary.     

Simple and rapid method determination for metformin in human plasma using high performance liquid chromatography tandem mass spectrometry: application to pharmacokinetic studies

A rapid and simple method for quantitation of metformin (MET) in human plasma by HPLC-MS/MS was developed and validated. The sample preparation consists of plasma deproteinization using acetonitrile. The mobile phase consisted of water-acetonitrile and formic acid (55/45/0.048, v/v/%) and the run time was 3 min. A pursuit C(18) (100 mm x 2.0 mm i.d., 3 microm) column connected to a guard column MS-pursuit (0.20 mm x 0.20 mm i.d., 5 microm) was used. The range of the calibration curve was from 20 to 5000 ng/mL, the limit of quantitation being 20 ng/mL. The detection was performed on a mass spectrometer (ESI+), using metoprolol as internal standard. The calibration curves have r(2) values of 0.995 (CV=0.24%, n=10). The accuracy and precision were between 90.74 and 106.7% and coefficients of variations (CV) of 1.10 and 4.35%, respectively. The method was applied to determine the pharmacokinetic parameters: C(max) (1667.25 ng/mL) and T(max) (3.89 h).     

Simultaneous determination of amodiaquine and its active metabolite in human blood by ion-pair liquid chromatography-tandem mass spectrometry

A sensitive and selective ion-pair liquid chromatography-tandem mass spectrometric method (IP-LC-MS/MS) for the simultaneous determination of amodiaquine (AQ) and its active metabolite, N-desethylamodiaquine (AQm), in human blood has been developed and validated. Pentafluoropropionic acid (PFPA) was applied as ion-pairing reagent in reversed-phase chromatographic separation. The effects of PFPA concentrations and the volume fraction of acetonitrile in the mobile phase on the retention of analytes were investigated on a Venusil MP-C(18) column, and the mobile phase was finally optimized as acetonitrile:water (23:77, v/v) with 0.0667% PFPA in the aqueous phase. The results proved that PFPA as an ion-pairing reagent could provide desirable chromatographic performance in the IP-LC-MS/MS determination of 4-aminoquinoline compounds. Blood samples were protein precipitated with acetonitrile using hydroxychloroquine (OHCQ) as the internal standard. The detection was carried out in multiple reaction monitoring (MRM) mode via positive atmospheric pressure chemical ionization (APCI) interface. The lower limits of quantification were established at 0.150 and 1.50 ng/mL for AQ and AQm, respectively. The validated IP-LC-MS/MS method was applied to a clinical pharmacokinetic study of AQ and AQm in human blood after an oral administration of 600 mg AQ hydrochloride (45 9mg base).     

Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis

Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of alpha-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (muRPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mug mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with muRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.     

Mechanical response and conformational changes of alpha-actinin domains during unfolding: a molecular dynamics study

Alpha-actinin is a cytoskeleton-binding protein involved in the assembly and regulation of the actin filaments. In this work molecular dynamics method was applied to investigate the mechanical behaviour of the human skeletal muscle α-actinin. Five configurations were unfolded at an elongation speed of 0.1 nm/ps in order to investigate the conformational changes occurring during the extension process. Moreover, a sensitivity analysis at different velocities was performed for one of the R2–R3 spectrin-like repeat configuration extracted in order to evaluate the effect of the pulling speed on the mechanical behaviour of the molecule. Two different behaviours were recognized with respect to the pulling speed. In particular, at speed higher than 0.025 nm/ps a continuous rearrangement without evident force peaks was obtained, on the contrary at lower speed evident peaks in the range 500–750 pN were detected. R3 repeat resulted more stable than R2 during mechanical unfolding, due to the lower hydrophobic surface available to the solvent. The characterization of the R2–R3 units can be useful for the development of cytoskeleton network models based on stiffness values obtained by analyses performed at the molecular level.     

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.