High-sensitivity TFA-free LC-MS for profiling histones

The analysis of proteins by RPLC commonly involves the use of TFA as an ion‐pairing agent, even though it forms adducts and suppresses sensitivity. The presence of adducts can complicate protein molecular weight assignment especially when protein isoforms coelute as in the case of histones. To mitigate the complicating effects of TFA adducts in protein LC‐MS, we have optimized TFA‐free methods for protein separation. Protein standards and histones were used to evaluate TFA‐free separations using capillary (0.3 mm id) and nanoscale (0.1 mm id) C₈ columns with the ion‐pairing agents, formic acid or acetic acid. The optimized method was then used to examine the applicability of the approach for histone characterization in human cancer cell lines and primary tumor cells from chronic lymphocytic leukemia patients.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment.

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Alkyl phosphotriester modified oligodeoxyribonucleotides. VI. NMR and UV spectroscopic studies of ethyl phosphotriester (Et) modified Rp-Rp and Sp-Sp duplexes, (d[GGAA(Et)TTCC])2.

1H NMR chemical shift assignments for the title compounds were made for all but a few H5' and H5" signals using two-dimensional nuclear Overhauser effect (2D-NOE) data, which was also used for the first time to assign absolute configuration at phosphorus. The chemical shifts were, in general, similar to those reported [Broido, M.S., et al. (1985) Eur. J. Biochem. 150, 117-128] for the B-like conformation of the unmodified, parent duplex, [d(GGAATTCC)]2. Differences in chemical shifts for corresponding protons were mostly localized to the AA(Et)TT region, and showed some stereochemical dependence. Unambiguous assignment of the phosphotriester 31P signals was achieved in a novel way using selective insensitive nucleus enhancement by polarization transfer (selective INEPT) NMR. The Rp-Rp duplex melted ca. 11 degrees C lower than either the Sp-Sp or parent duplexes, as evidenced by Tm and variable temperature 1H/31P NMR measurements. The 2D-NOE data for the Rp-Rp duplex suggested possible steric interactions between the ethyl group and the H3' of the flanking A residue. At low ionic strength, the Sp-Sp and parent duplexes had similar stability but at high ionic strength the Sp-Sp duplex was less stable.     

Quantification of valproic acid and its metabolite 2-propyl-4-pentenoic acid in human plasma using HPLC-MS/MS

A specific and sensitive HPLC-MS/MS method for the quantitative determination of valproic acid (VPA) and its metabolite, 2-propyl-4-pentenoic acid in human plasma has been developed, using VPA-d15 as the internal standard. The method was based on pre-column derivatization using 4-dimethylaminobenzylamine dihydrochloride. The derivatives were separated with a gradient elution and quantified by positive electrospray ionization with multiple reaction monitoring. The assay provides routine quantification limits of 200 ng/mL for VPA and 20 ng/mL for 4-ene VPA with within- and between-day coefficients of variation of <10%. This method has been applied to the analysis of plasma samples obtained from patients treated with this drug.     

XIAP antisense oligonucleotide (AEG35156) achieves target knockdown and induces apoptosis preferentially in CD34<Superscript>+</Superscript>38<Superscript>−</Superscript> cells in a phase 1/2 study of patients with relapsed/refractory AML

XIAP, a potent caspase inhibitor, is highly expressed in acute myeloid leukemia (AML) cells and contributes to chemoresistance. A multi-center phase 1/2 trial of XIAP antisense oligonucleotide AEG35156 in combination with idarubicin/cytarabine was conducted in 56 patients with relapsed/refractory AML. Herein we report the pharmacodynamic studies of the patients enrolled at M. D. Anderson Cancer Center. A total of 13 patients were enrolled in our institution: five in phase 1 (12–350 mg/m² AEG35156) and eight in phase 2 (350 mg/m² AEG35156) of the protocol. AEG35156 was administered on 3 consecutive days and then weekly up to a maximum of 35 days. Blood samples were collected from patients on days 1 through 5 and on day 28–35 post-chemotherapy for detection of XIAP levels and apoptosis. AEG35156 treatment led to dose-dependent decreases of XIAP mRNA levels (42–100% reduction in phase 2 patients). XIAP protein levels were reduced in all five samples measured. Apoptosis induction was detected in 1/4 phase 1 and 4/5 phase 2 patients. Importantly, apoptosis was most pronounced in CD34 ⁺ 38 ⁻ AML stem cells and all phase 2 patients showing apoptosis induction in CD34 ⁺ 38 ⁻ cells achieved response. We conclude that at 350 mg/m², AEG35156 is effective in knocking down XIAP in circulating blasts accompanied by the preferential induction of apoptosis in CD34 ⁺ 38 ⁻ AML stem cells.     

Tag retention and survival of juvenile bighead carp implanted with a dummy acoustic tag at three temperatures

Bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix (together, the bigheaded carps) are invasive fishes in North America that have resulted in substantial negative effects on native fish communities and aquatic ecosystems. Movement and behavior of adult bigheaded carps has been studied previously using telemetry, while similar studies with juvenile bigheaded carps have yet to be attempted. Recent technological advances in telemetry transmitters has increased the availability of tags sufficiently small enough to implant in juvenile carps. However, the effects of surgical implantation of telemetry tags on juvenile bigheaded carps have not been evaluated. We determined tag retention and survival associated with surgical implantation of acoustic telemetry tags into juvenile bighead carp (range 128–152 mm total length) at three temperatures (13, 18, and 23°C). In addition, we assessed the effect of surgically implanted transmitters on the fitness, defined as changes in weight or critical swimming speed, of carp implanted with transmitters. Survival was high among tagged fish (85%) with 47% of tags retained at the conclusion of the 45‐day study. No substantial decline in fitness of the fish was observed in tagged fish compared to untagged fish.     

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : I. CO(2) Assimilation and Metabolism and Activities of Phosphoenolpyruvate Carboxylase and NADP-Malic Enzyme

The degree of C₄ photosynthesis was assessed in four hybrids among C₄, C₄-like, and C₃-C₄ species in the genus Flaveria using ¹⁴C labeling, CO₂ exchange, ¹³C discrimination, and C₄ enzyme activities. The hybrids incorporated from 57 to 88% of the ¹⁴C assimilated in a 10-s exposure into C₄ acids compared with 26% for the C₃-C₄ species Flaveria linearis, 91% for the C₄ species Flaveria trinervia, and 87% for the C₄-like Flaveria brownii. Those plants with high percentages of ¹⁴C initially fixed into C₄ acids also metabolized the C₄ acids quickly, and the percentage of ¹⁴C in 3-phosphoglyceric acid plus sugar phosphates increased for at least a 30-s exposure to ¹²CO₂. This indicated a high degree of coordination between the carbon accumulation and reduction phases of the C₄ and C₃ cycles. Synthesis and metabolism of C₄ acids by the species and their hybrids were highly and linearly correlated with discrimination against ¹³C. The relationship of ¹³C discrimination or ¹⁴C metabolism to O₂ inhibition of photosynthesis was curvilinear, changing more rapidly at C₄-like values of ¹⁴C metabolism and ¹³C discrimination. Incorporation of initial ¹⁴C into C₄ acids showed a biphasic increase with increased activities of phosphoenolpyruvate carboxylase and NADP-malic enzyme (steep at low activities), but turnover of C₄ acids was linearly related to NADP-malic enzyme activity. Several other traits were closely related to the in vitro activity of NADP-malic enzyme but not phosphoenolpyruvate carboxylase. The data indicate that the hybrids have variable degrees of C₄ photosynthesis but that the carbon accumulation and reduction portions of the C₄ and C₃ cycles are well coordinated.