Sensitive liquid chromatography-mass spectrometry assay for quantitation of docetaxel and paclitaxel in human plasma

We have developed a high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS) method for quantifying docetaxel and paclitaxel in human plasma. The assay fulfills the need for defining the lower plasma concentrations of these antineoplastic agents that result from a number of changes in how these agents are used clinically. The assay uses paclitaxel as the internal standard for docetaxel, and vice versa; solid-phase extraction; a Phenomenex Hypersil ODS (5 micrometer, 100x2 mm) reversed-phase analytical column; an isocratic mobile phase of 0.1% formic acid in methanol-water (70:30, v/v); and mass spectrometric detection using electrospray positive mode electron ionization. The assay has a lower limit of quantitation (LLOQ) of 0.3 nM and is linear between 0.3 nM and 1 microM for docetaxel. For paclitaxel, the LLOQ was 1 nM, and the assay is linear between 1 nM and 1 microM. We demonstrated the suitability of this assay for docetaxel by using it to quantify the docetaxel concentrations in plasma of a patient given 40 mg/m(2) of docetaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. In a similar manner, the suitability of the assay for paclitaxel was demonstrated by using it to quantify the concentrations of paclitaxel in the plasma of a patient given 15 mg/m(2) of paclitaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. The LC-MS assay, which proved superior because of its greater sensitivity and relatively short (7 min) run time, should be an important tool for future pharmacokinetic analyses of docetaxel and paclitaxel.     

Ascorbic acid and alpha-tocopherol as potent modulators on arsenic induced toxicity in mitochondria

Arsenic exists ubiquitously in our environment and various forms of arsenic circulate in air, water, soil and living organisms. Since arsenic compounds have shown to exert their toxicity chiefly by generating reactive oxygen species, we have evaluated the effect of antioxidants ascorbic acid and alpha-tocopherol on lipid peroxidation, antioxidants and mitochondrial enzymes in liver and kidney of arsenic exposed rats. A significant increase in the level of lipid peroxidation and decrease in the levels of antioxidants and in the activities of mitochondrial enzymes were observed in arsenic intoxicated rats. Co-administration of arsenic treated rats with ascorbic acid and alpha-tocopherol showed significant reduction in the level of lipid peroxidation and elevation in the levels of ascorbic acid, alpha-tocopherol, glutathione and total sulfhydryls and in the activities of isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, NADH-dehydrogenase and cytochrome c oxidase. From our results, we conclude that ascorbic acid and alpha-tocopherol alleviate arsenic- induced alterations in mitochondria.     

What Is the Signal for the Posttranslational Arginylation of Proteins?

The N-terminal, posttranslational arginylation of proteins is ubiquitous in eukaryotic cells. Previous experiments, using purified components of the reaction incubated in the presence of exogenous substrates, have shown that only those proteins containing acidic residues at their N-terminals are arginylation substrates. However, data from experiments that used crude extracts of brain and nerve as the source of the arginylating molecules, suggest that the in vivo targets for arginylation are more complex than those demonstrated using purified components. One of the proposed functions for arginylation is as a signal for protein degradation and proteins that have undergone oxidative damage have been shown to be rapidly degraded. In the present experiments we have tested the hypothesis that the presence of an oxidatively damaged residue in a protein is a signal for its arginylation. These experiments have been performed by adding synthetic oxidized peptides to crude extracts of rat brain, incubating them with [³H]Arg and ATP and assaying for arginylated peptides using RP-HPLC. Results showed that while the oxidized A-chain of insulin was arginylated in this system, confirming previous experiments, other peptides containing oxidized residues were not. When a peptide containing Glu in the N-terminus was incubated under the same conditions it too was not a substrate for arginylation. These findings show that neither the presence of an N-terminal acidic residue nor an oxidized residue alone are sufficient to signal arginylation. Thus, another feature of the oxidized A-chain of insulin is required for arginylation. That feature remains to be identified.     

Sleep-Dependent Reactivation of Ensembles in Motor Cortex Promotes Skill Consolidation

Despite many prior studies demonstrating offline behavioral gains in motor skills after sleep, the underlying neural mechanisms remain poorly understood. To investigate the neurophysiological basis for offline gains, we performed single-unit recordings in motor cortex as rats learned a skilled upper-limb task. We found that sleep improved movement speed with preservation of accuracy. These offline improvements were linked to both replay of task-related ensembles during non-rapid eye movement (NREM) sleep and temporal shifts that more tightly bound motor cortical ensembles to movements; such offline gains and temporal shifts were not evident with sleep restriction. Interestingly, replay was linked to the coincidence of slow-wave events and bursts of spindle activity. Neurons that experienced the most consistent replay also underwent the most significant temporal shift and binding to the motor task. Significantly, replay and the associated performance gains after sleep only occurred when animals first learned the skill; continued practice during later stages of learning (i.e., after motor kinematics had stabilized) did not show evidence of replay. Our results highlight how replay of synchronous neural activity during sleep mediates large-scale neural plasticity and stabilizes kinematics during early motor learning.     

Energetic Calculations to Decipher pH-Dependent Oligomerization and Domain Swapping of Proteins

Domain swapping mechanism is a specialised mode of oligomerization of proteins in which part of a protein is exchanged in a non-covalent manner between constituent subunits. This mechanism is highly affected by several physiological conditions. Here, we present a detailed analysis ofthe effect of pH on different regions of the domain swapped oligomer by considering examples which are known to be sensitive to pH in transiting from monomeric to domain-swapped dimeric form. The energetic calculations were performed using a specialized method which considers changes in pH and subsequent changes in the interactions between subunits. This analysis provides definitive hints about the pH-dependence switch from monomer to domain-swapped oligomer and the steps that may be involved in the swapping mechanism.     

Expression,purification and substrate specificities of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2

3-Nitotoluene dioxygenase (3-NTDO) is the first enzyme in the degradation pathway of 3-nitrotoluene (3-NT) by Diaphorobacter sp. strain DS2. The complete gene sequences of 3-NTDO were PCR amplified from genomic DNA of Diaphorobacter sp., cloned, sequenced and expressed. The 3-NTDO gene revealed a multi component structure having a reductase, a ferredoxin and two oxygenase subunits. Clones expressing the different subunits were constructed in pET21a expression vector system and overexpressed in E. coli BL21(DE3) host. Each subunit was individually purified separately to homogeneity. The active recombinant enzyme was reconstituted in vitro by mixing all three purified subunits. The reconstituted recombinant enzyme could catalyse biotransformations on a variety of organic aromatics.     

Effect of breed and sperm concentration on the changes in structural, functional and motility parameters of ram-lamb spermatozoa during storage at 4 degrees C

The objectives of this study were (1) to determine the changes in structural, functional and motility parameters of ram-lamb semen stored at two different concentrations at 4 degrees C for 8 days in egg-yolk based extender and (2) to determine the effect of breed of ram-lambs on the changes in structural, functional and motility parameters of ram-lamb semen from different breeds stored at two different concentrations at 4 degrees C for 8 days in egg-yolk based extender. Two different concentrations suitable for laparoscopic and cervical insemination were employed in this experiment. A total of 14 ram-lambs (Polled Dorset-5, Suffolk-5, Katahdin-4) with satisfactory breeding potential were selected. Semen samples were collected by electro-ejaculation. Semen samples were extended to 50 and 200 million sperm per ml with a commercial egg yolk based extender (Triladyl, Minitube of America, Verona, WI, USA) at room temperature and were stored at 4 degrees C. The sperm DNA fragmentation index (DFI), percentages of high mitochondrial membrane potential (hMMP) and plasma membrane integrity (PMI) were assessed using flow cytometry as part of structural and functional parameters on Days 0, 1, 4, 6, and 8. A computer assisted sperm analyser (HTM-IVOS, Version 10.8, Hamilton Thorne Research, Beverly, MA, USA) was used to assess the sperm motility parameters on Days 0, 1, 4, 6, and 8. PROC MIXED procedure was used to determine the effect of days of storage, concentration and breed. The concentration and days of storage significantly affected the sperm structural, functional and motility parameters (P<0.0001). Significant concentration x days of storage interaction was found for all structural and functional parameters. There was a significant concentration x days of storage interaction for average path velocity, curvilinear velocity, straightness and linearity. Overall changes in the sperm structural, functional and sperm motility parameters over the storage period were less dramatic in the 200 x 10(6) ml(-1) concentration when compared to 50 x 10(6) ml(-1) concentration. The hMMP and total progressive motility were influenced by breed. In conclusion, the quality of structural, functional and motility parameters declined as days of storage were increased and the magnitude of changes in the parameters was less dramatic at the higher concentration.     

Semantic integration to identify overlapping functional modules in protein interaction networks

Background  

The systematic analysis of protein-protein interactions can enable a better understanding of cellular organization, processes and functions. Functional modules can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of functional module detection algorithms.     

Checkpoint independence of most DNA replication origins in fission yeast

Background

In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2).

Results

Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.

Conclusion

The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.