Simultaneous quantification of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS)

Cyclophosphamide is a cytotoxic prodrug with a very narrow therapeutic index. To study the clinical pharmacology of cyclophosphamide in a large cohort of patients a previously published method for the simultaneous quantitative determination of cyclophosphamide and 4-hydroxycyclophosphamide in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS) was optimized. Addition of an isotopically labelled internal standard and adaptation of the gradient resulted in a fast, robust and sensitive assay. Because 4-hydroxycyclophosphamide is not stable in plasma, the compound is derivatized with semicarbazide immediately after sample collection. Sample preparation was carried out by protein precipitation with methanol-acetonitrile (1:1, v/v), containing isotopically labelled cyclophosphamide and hexamethylphosphoramide as internal standards. The LC separation was performed on a Zorbax Extend C18 column (150 mm x 2.1 mm ID, particle size 5 microm) with 1 mM ammonium hydroxide in water-acetonitrile (90:10, v/v) as the starting gradient, at a flow-rate of 0.40 mL/min with a total run time of 6 min. The lower limit of quantification (LLQ, using a 100 microL sample volume) was 200 ng/mL and the linear dynamic range extended to 40,000 ng/mL for cyclophosphamide and 50-5000 ng/mL for 4-hydroxycyclophosphamide. Accuracies as well as precisions were lower than 20% at the LLQ concentration and lower than 15% for all other concentrations. This method has been successfully applied in our institute to support ongoing studies into the pharmacokinetics and pharmacogenetics of cyclophosphamide.     

A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana

Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.     

CD4<Superscript>+</Superscript> T cells kill Id<Superscript>+</Superscript> B-lymphoma cells: FasLigand-Fas interaction is dominant in vitro but is redundant in vivo

B-lymphoma cells express a highly tumor-specific antigen, monoclonal Ig, which is a promising target for immunotherapy. Previous work has demonstrated that B-lymphoma cells spontaneously process their endogenous monoclonal Ig and present variable (V) region peptides (Id-peptides) on their MHC class II molecules to CD4⁺ T cells. Id-specific CD4⁺ T cells protect mice against B-lymphoma cells in the absence of anti-idiotypic antibodies. The molecular mechanism by which Id-specific CD4⁺ T cells kill B-lymphoma cells is hitherto unknown. We here demonstrate in an Id-specific T-cell receptor (TCR)–transgenic mouse model that Id-specific CD4⁺ T cells induce apoptosis of Fas⁺ B-lymphoma cells in vitro by FasLigand (FasL)–Fas interaction. Moreover, the rare B lymphomas that had escaped rejection in TCR-transgenic mice had down-regulated their sensitivity to Fas-mediated apoptosis. Although these results suggest that FasL-Fas interaction is important, Id-specific CD4⁺ T cells could eliminate Id⁺ B-lymphoma cells in vivo by other mechanisms, since three independent ways of blocking FasL-Fas–mediated killing failed to abrogate tumor protection in TCR-transgenic mice. These results suggest that there are several redundant pathways by which Id-specific CD4⁺ T cells eliminate Id⁺ B-lymphoma cells in vivo, of which FasL-Fas interaction is only one.Supported by grants from the Norwegian Cancer Society, the Research Council of Norway, and the Multiple Myeloma Research Foundation.     

Anti-complement effects of lactoferrin-derived peptides

Lactoferrin is an important biological molecule with many functions such as modulation of the inflammatory response, iron metabolism and antimicrobial defense. One effect of lactoferrin is the inhibition of the classical complement pathway. This study reports that antimicrobial peptides derived from the N-terminal region from both human and bovine lactoferrin, lactoferricin H and lactoferricin B, respectively, inhibit the classical complement pathway. No inhibitory effect of these peptides was observed on the alternative complement pathway in an AP50 assay. However, lactoferricin B reduced the inhibitory properties of serum against Escherichia coli in a concentration dependent manner. These results suggest that the N-terminal region of lactoferrin is the important part in the inhibition of complement activation and that these peptides possess other important properties than their antimicrobial effect.     

Multiple resonances among time structures, chronomes, around and in US. Is an about 1.3-year periodicity in solar wind built into the human cardiovascular chronome?

AIMS: Velocity changes in the solar wind, recorded by satellite (IMP8 and Wind) are characterized by a solar cycle dependent approximately 1.3-year component. The presence of any approximately 1.3-year component in human blood pressure (BP) and heart rate (HR) and in mortality from myocardial infarction (MI) is tested and its relative prominence compared to the 1.0-year variation. MATERIALS AND METHODS: Around the clock manual or automatic BP and HR measurements from four subjects recorded over 5 to 35 years and a 29-year record of mortality from MI in Minnesota were analyzed by linear-nonlinear rhythmometry. Point and 95% confidence interval (CI) estimates were obtained for the approximately 1.3-year period and amplitude. The latter is compared with the 1.0-year amplitude for BP and HR records concurrent to the solar data provided by one of us (JDR). RESULTS: An approximately 1.3-year component is resolved nonlinearly for MI, with a period of 1.23 (95% CI: 1.21; 1.26) year. This component was invariably validated with statistical significance for BP and HR by linear rhythmometry. Nonlinearly, the 95% CI for the 1.3-year amplitude did not overlap zero in 11 of the 12 BP and HR series. Given the usually strong synchronizing role of light and temperature, it is surprising that 5 of the 12 cardiovascular series had a numerically larger amplitude of the 1.3-year versus the precise 1.0-year component. The beating of the approximately 1.3-year and 1.0-year components was shown by gliding spectra on actual and simulated data. DISCUSSION AND CONCLUSION: The shortest 5-year record (1998-2003) revealed an approximately 1.3-year component closer to the solar wind speed period characterizing the entire available record (1994-2003) than that for the concurrent 5-year span. Physiological variables may resonate with non-photic environmental cycles that may have entered the genetic code during evolution.     

A revised annotation and comparative analysis of Helicobacter pylori genomes

Huge amounts of genomic information are currently being generated. Therefore, biologists require structured, exhaustive and comparative databases. The PyloriGene database (http://genolist.pasteur.fr/PyloriGene) was developed to respond to these needs, by integrating and connecting the information generated during the sequencing of two distinct strains of Helicobacter pylori. This led to the need for a general annotation consensus, as the physical and functional annotations of the two strains differed significantly in some cases. A revised functional classification system was created to accommodate the existing data and to make it possible to classify coding sequences (CDS) into several functional categories to harmonize CDS classification. The annotation of the two complete genomes was revised in the light of new data, allowing us to reduce the percentage of hypothetical proteins from approximately 40 to 33%. This resulted in the reassignment of functions for 108 CDS (approximately 7% of all CDS). Interestingly, the functions of only approximately 13% of CDS (222 out of 1658 CDS) were annotated as a result of work done directly on H.pylori genes. Finally, comparison of the two published genomes revealed a significant amount of size variation between corresponding (orthologous) CDS. Most of these size variations were due to natural polymorphisms, although other sources of variation were identified, such as pseudogenes, new genes potentially regulated by slipped-strand mispairing mechanism, or frame-shifts. 113 of these differences were due to different start codon assignments, a common problem when constructing physical annotations.     

Competitive inhibition by Rauber's sickle of the primitive streak and/or (pre)neural plate inducing effects of sickle endoblast in avian blastoderms

When in unincubated chicken blastoderms the Rauber's sickle is (sub)totally mechanically removed by selective scraping, the further evolution of the blastoderm in culture is often profoundly disturbed, going from only expansion of the upper layer and preneural plate formation to the development of a slowly growing miniature embryo. Our results suggest that the developmental potencies of the embryo are related to the presence or absence of Rauber's sickle material left after its removal. This can be checked after culture by the presence or nonpresence of junctional endoblast (derived from Rauber's sickle) and the concomitant induction of blood islands in the immediate neighborhood. Our study thus indicates that without Rauber's sickle (in the cases of successful total selective removal), an avian blastoderm cannot develop normally, even in the presence of an intact caudal marginal zone. After placing a fragment of quail sickle endoblast on the anti-sickle region of unincubated chicken blastoderms from which the Rauber's sickle was (sub)totally removed, different developmental scenarios were seen, according to the degree of removal, both in the anti-sickle as in the sickle regions. 1) If Rauber's sickle activity is strongly reduced, then besides a centripetally directed miniature embryo, induced by the remnants of the autochthonous Rauber's sickle, an additional centripetally directed embryo or preneural plate (without accompanying blood islands) develops in the anti-sickle region under inductory influence of the apposed quail sickle endoblast. We make a distinction between a neural plate and a preneural plate. The latter consists of a thickening of the upper layer (with the same initial aspect as a neural plate) adjacent to endophyll or sickle endoblast in the absence of chordomesoblast and gastrulation phenomena. 2) If Rauber's sickle activity is totally absent, then the inducing power of the sickle endoblast fragment becomes maximal and, starting from the anti-sickle region, one single embryo (without blood islands) extending over the whole area centralis appears. 3) If much of the Rauber's sickle material has been left in the blastoderm, then the inducing activity of the sickle endoblast, placed on the anti-sickle region, will be totally suppressed (although the sickle endoblast remains intact) and neither a preneural plate nor a primitive streak was induced. After placing a fragment of quail sickle endoblast on the anti-sickle region of an unincubated chicken blastoderm from which the Rauber's sickle and surrounding tissues were completely excised, an embryo was always induced by the sickle endoblast in the adjacent upper layer of this anti-sickle region. In the absence of sickle endoblast, this never occurred. Thus, our experiments demonstrate that in the absence of the Rauber's sickle, a parent tissue (sickle endoblast) induces both gastrulation and neurulation phenomena, while in the full presence of Rauber's sickle these functions are totally suppressed. Moreover, Rauber's sickle not only organizes gastrulation and blood island formation by itself but also influences neurulation at a distance (in space and time) by part of its cell lineage (i.e., sickle endoblast). Our study suggests that the inhibitory effect of Rauber's sickle on its parent tissue (sickle endoblast) represents an early mechanism impairing polyembryony, so that only a single primary major organizer (Rauber's sickle) remains active in the young avian germinal disc.     

Combined spatial and enzymatic regulation of Csk by cAMP and protein kinase a inhibits T cell receptor signaling

Raft-associated Csk controls signaling through the T cell receptor (TCR) and was mainly anchored to Cbp/PAG (phosphoprotein associated with glycosphingolipid-enriched membrane domains). Treatment of cells with the cAMP-elevating agent prostaglandin E(2) (PGE(2)) augmented the level of Cbp/PAG phosphorylation with a concomitant increase in amounts of Csk bound to Cbp/PAG. While TCR-triggering resulted in transient dissociation of Csk from Cbp/PAG/rafts allowing TCR-induced tyrosine phosphorylation to occur, pretreatment with PGE(2) reduced Csk dissociation upon TCR triggering. This correlated with lowered TCR-induced phosphorylation of CD3 zeta-chain and linker for activation of T cells. Moreover, competition of endogenous Csk from lipid rafts abolished PGE(2)-mediated inhibition of TCR-induced zeta-chain phosphorylation and activation of the nuclear factor of activated T cells (NFAT) activator protein 1 (AP-1). Finally, raft-associated Csk already activated via Cbp/PAG binding, gained additional increase in phosphotransferase activity upon protein kinase A-mediated phosphorylation of Csk. We propose that cAMP regulates Csk via both spatial and enzymatic mechanisms, thereby inhibiting signaling through the TCR.     

How well can blood pressure be controlled? Progress report on the Systolic Hypertension in Europe Follow-Up Study (Syst-Eur 2)

Background

The randomised, double-blind, placebo-controlled Systolic Hypertension in Europe trial (Syst-Eur 1) proved that blood pressure (BP) lowering therapy starting with nitrendipine reduces the risk of cardiovascular complications in elderly patients with isolated systolic hypertension. In an attempt to confirm the safety of long-term antihypertensive therapy based on a dihydropyridine, the Syst-Eur patients remained in open follow-up after the end of Syst-Eur 1. This paper presents the second progress report of this follow-up study (Syst-Eur 2). It describes BP control and adherence to study medications.

Methods

After the end of Syst-Eur 1 all patients, treated either actively or with placebo, were invited either to continue or to start antihypertensive treatment with the same drugs as previously used in the active treatment arm. In order to reach the target BP (sitting SBP <150 mmHg), the first line agent, nitrendipine, could be associated with enalapril and/or hydrochlorothiazide.

Results

Of the 3787 eligible patients, 3516 (93%) entered Syst-Eur 2. At the last available visit, 72% of the patients were taking nitrendipine. SBP/DBP at entry in Syst-Eur 2 averaged 160/83 mmHg in the former placebo group and 151/80 mmHg in the former active-treatment group. At the last follow-up visit SBP/DBP in the patients previously randomised to placebo or active treatment had decreased by 16/5 mmHg and 7/5 mmHg, respectively. The target BP was reached by 74% of the patients.

Conclusion

Substantial reductions in systolic BP may be achieved in older patients with isolated systolic hypertension with a treatment strategy starting with the dihydropyridine calcium-channel blocker, nitrendipine, with the possible addition of enalapril and/or hydrochlorothiazide.     

In the absence of Rauber's sickle material,no blood islands are formed in the avian blastoderm

Using the quail-chick chimera technique, we followed the fate of Rauber's sickle cells in older whole blastoderms (cultured for approximately 2 days): after removal of the autochthonous Rauber's sickle from an unincubated chicken blastoderm, a quail Rauber's sickle was grafted isotopically and isochronically in its place. In transverse sections through these chimeras, the grafted quail Rauber's sickle cells were seen to have transformed into a broad row or ridge of quail junctional endoblast cells extending at the inner border of the area containing blood islands. After unilateral removal of the junctional endoblast from an intermediate streak chicken blastoderm (Stage 3; Hamburger and Hamilton [1951] J Morphol 88:49-92), we observed during further in vitro culture that at the operated side, in the area previously occupied by this junctional endoblast, blood islands no longer developed. If after such a unilateral removal of the chicken junctional endoblast quail junctional endoblast was apposed in its place, then blood islands reappeared in the operated area. The intimate contact between the apposed quail junctional endoblast and the recently formed blood islands, derived from peripherally migrating mesoderm, was very obvious on sections through such chimeras. We further demonstrate that Rauber's sickle vs. junctional endoblast is indispensable for the anlage of blood islands in avian blastoderms. Indeed, in the absence of Rauber's sickle material no blood islands develop (even when mesoderm is present after ingression of the upper layer via a primitive streak) in the isolated central region of the area centralis of unincubated chicken blastoderms after culture in vitro. Also, no junctional endoblast and no sickle canal appear in these explants. By contrast, if a Rauber's sickle fragment is placed on such an isolated central blastoderm region, then blood islands develop. These blood islands start to develop from peripherally migrating mesoderm in the neighborhood of the Rauber's sickle-derived junctional endoblast.