Detection of nonsterol isoprenoids by HPLC-MS/MS

Isoprenoids constitute an important class of biomolecules that participate in many different cellular processes. Most available detection methods allow the identification of only one or two specific nonsterol isoprenoid intermediates following radioactive or fluorescent labeling. We here report a rapid, nonradioactive, and sensitive procedure for the simultaneous detection and quantification of the eight main nonsterol intermediates of the isoprenoid biosynthesis pathway by means of tandem mass spectrometry. Intermediates were analyzed by HPLC-MS/MS in the multiple reaction monitoring mode using a silica-based C₁₈ HPLC column. For quantification, their stable isotope-labeled analogs were used as internal standards. HepG2 cells were used to validate the method. Mevalonate, phosphomevalonate, and the six subsequent isoprenoid pyrophosphates were readily determined with detection limits ranging from 0.03 to 1.0 μmol/L. The intra- and interassay variations for HepG2 cell homogenates supplemented with isoprenoid intermediates were 3.6-10.9 and 4.4-11.9%, respectively. Under normal culturing conditions, isoprenoid intermediates in HepG2 cells were below detection limits. However, incubation of the cells with pamidronate, an inhibitor of farnesyl pyrophosphate synthase, resulted in increased levels of mevalonate, isopentenyl pyrophosphate/dimethylallyl pyrophosphate, and geranyl pyrophosphate. This method will be suitable for measuring profiles of isoprenoid intermediates in cells with compromised isoprenoid biosynthesis and for determining the specificity of potential inhibitors of the pathway.     

Distinct glycoforms of human α1-acid glycoprotein have comparable synthesis rates: a [13C]valine-labelling study in healthy humans

Various α₁-acid glycoprotein (AGP) glycoforms are present in plasma differing in extent of branching and/or fucosylation of their 5 N-linked glycans, as well as in concentration. It is assumed that hepatic synthesis determines the relative occurrence of the AGP-glycoforms in plasma, but experimental evidence is lacking. In this study, we have investigated the contribution of fractional synthesis rates to the plasma concentration of AGP-glycoforms that differed in relative occurrence in healthy human plasma. During a [¹³C]valine infusion, AGP was isolated from the plasma of healthy volunteers. Four AGP-glycoforms, differing strongly in plasma concentration were obtained by sequential affinity chromatography over concanavalin-A- and Aleuria aurantia-agarose columns. The incorporation of the [¹³C]valine tracer into the AGP-glycoforms was measured by gas chromatography combustion isotope ratio mass spectrometry. The mean fractional synthesis rates of the four AGP-glycoforms did not differ significantly between each other as well between individuals. The results indicated a renewal of about 15%/day of the plasma pools of each of the AGP-glycoforms. This is in support to the assumption that the differences in plasma concentration of the AGP-glycoforms are a reflection of the state of the hepatic glycosylation process. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of the Bcl-2 Protein BAD Promotes Prostate Cancer Growth

BAD, a pro-apoptotic protein of the Bcl-2 family, has recently been identified as an integrator of several anti-apoptotic signaling pathways in prostate cancer cells. Thus, activation of EGFR, GPCRs or PI3K pathway leads to BAD phosphorylation and inhibition of apoptosis. Increased levels of BAD in prostate carcinomas have also been reported. It appears contradictory that instead of limiting expression of pro-apoptotic protein, prostate cancer cells choose to increase BAD levels while keeping it under tight phosphorylation control. Analysis of the effect of BAD on prostate cancer xenografts has shown that increased BAD expression enhances tumor growth, while knockdown of BAD expression by shRNA inhibits tumor growth. Tissue culture experiments demonstrated that increased BAD expression stimulates proliferation of prostate cancer cells. These results suggest that increased expression of BAD provides a proliferative advantage to prostate tumors, while BAD dephosphorylation increases sensitivity of prostate cancer cells to apoptosis. Combination of proliferative and apoptotic properties prompts prostate cancer cells to be “addicted” to increased levels of phosphorylated BAD. Thus, kinases that phosphorylate BAD are plausible therapeutic targets; while monitoring BAD phosphorylation could be used to predict tumor response to treatments.     

Detection ofFusarium tricinctum from cereal grain using PCR assay

Contamination of cereals with mycotoxins produced by Fusarium is a worldwide problem requiring rapid and sensitive detection methods. This paper describes the development of a PCR protocol facilitating the detection of F. tricinctum, which belongs to the FHB (Fusarium Head Blight) complex responsible for contamination of cereal grains with enniatins and moniliformin. Sequence alignment of partial IGS rDNA revealed a single nucleotide polymorphism, which was used to design primers differentiating F. tricinctum from other members of the FHB complex. The specificity of the assay was tested on 68 isolates belonging to 21 Fusarium species originating from different parts of the world and hosts/substrates. Positive PCR results were obtained from all 12 F. tricinctum isolates tested; however, unexpected amplicons were amplified from the templates of F. acuminatum (CBS 618.87) and F. nurragi (CBS 393.96). No cross reactivity was found with any other Fusarium species tested. The PCR assay was tested on 24 asymptomatic wheat seed samples originating from Northern Poland and resulted in 13 positive samples, of which 11 samples were contaminated with moniliformin and/or antibiotic Y.     

Nanodomain coupling between Ca2+ channels and Ca2+ sensors promotes fast and efficient transmitter release at a cortical GABAergic synapse

It is generally thought that transmitter release at mammalian central synapses is triggered by Ca2+ microdomains, implying loose coupling between presynaptic Ca2+ channels and Ca2+ sensors of exocytosis. Here we show that Ca2+ channel subunit immunoreactivity is highly concentrated in the active zone of GABAergic presynaptic terminals of putative parvalbumin-containing basket cells in the hippocampus. Paired recording combined with presynaptic patch pipette perfusion revealed that GABA release at basket cell-granule cell synapses is sensitive to millimolar concentrations of the fast Ca2+ chelator BAPTA but insensitive to the slow Ca2+ chelator EGTA. These results show that Ca2+ source and Ca2+ sensor are tightly coupled at this synapse, with distances in the range of 10-20 nm. Models of Ca2+ inflow-exocytosis coupling further reveal that the tightness of coupling increases efficacy, speed, and temporal precision of transmitter release. Thus, tight coupling contributes to fast feedforward and feedback inhibition in the hippocampal network.     

Absolute versus per unit body length speed of prey as an estimator of vulnerability to predation

To study whether absolute (m/s) or relative (body lengths/s) speed should be used to compare the vulnerability of differently sized animals, we developed a simple computer simulation. Human 'predators' were asked to 'catch' (mouse-click) prey of different sizes, moving at different speeds across a computer screen. Using the simulation, a prey's chances of escaping predation depended on its speed (faster prey were more difficult to catch than slower prey of the same body size), but also on its size (larger prey were easier to catch than smaller prey at the same speed). Catching time, the time needed to catch a prey, also depended on both prey speed and prey size. Relative prey speed (body lengths/s or body surface/s) was a better predictor of catching time than was absolute prey speed (m/s). Our experiment demonstrates that, in contrast to earlier assertions, per unit body length speed of prey may be more 'ecologically relevant' than absolute speed. Copyright 1998 The Association for the Study of Animal Behaviour.     

On the role of alphaThr183 in the allosteric regulation and catalytic mechanism of tryptophan synthase

The catalytic activity and substrate channeling of the pyridoxal 5'-phosphate-dependent tryptophan synthase alpha(2)beta(2) complex is regulated by allosteric interactions that modulate the switching of the enzyme between open, low activity and closed, high activity states during the catalytic cycle. The highly conserved alphaThr183 residue is part of loop alphaL6 and is located next to the alpha-active site and forms part of the alpha-beta subunit interface. The role of the interactions of alphaThr183 in alpha-site catalysis and allosteric regulation was investigated by analyzing the kinetics and crystal structures of the isosteric mutant alphaThr183Val. The mutant displays strongly impaired allosteric alpha-beta communication, and the catalytic activity of the alpha-reaction is reduced one hundred fold, whereas the beta-activity is not affected. The structural work establishes that the basis for the missing inter-subunit signaling is the lack of loop alphaL6 closure even in the presence of the alpha-subunit ligands, 3-indolyl-D-glycerol 3'-phosphate, or 3-indolylpropanol 3'-phosphate. The structural basis for the reduced alpha-activity has its origins in the missing hydrogen bond between alphaThr183 and the catalytic residue, alphaAsp60.     

Influence of tumor drug resistance phenotype on the dynamics of cisplatin-induced changes of rat peripheral lymphocyte chromatin structure in Guerin's carcinoma

The correlation between the tumor drug sensitivity and the degree of lymphocyte interphase nuclei chromatin damages induced by cisplatin in rats was found using sensitive and resistant to cisplatin variants of Guerin's carcinoma. Increased optical density of lymphocyte chromatin in first minutes after cisplatin injection both in rats without Guerin's carcinoma and with sensitive to cisplatin variants of this tumor was observed. Lymphocyte chromatin structure remains unchanged in rats with cisplatin resistant carcinoma. Normal blood cells are suppose to change their sensitiveness to cisplatin under the humoral influence of the growing tumor in according with its phenotype.     

The effects of fellow patients on the emotional well-being and satisfaction with care of postoperative cosmetic surgery patients

This article reports the findings of a quasi-experimental study that represents the first attempt to systematically examine the possibility that contact with fellow patients after cosmetic surgery significantly influences a patient's postoperative emotional well-being and satisfaction with care. Patients were assigned to rooms that either facilitated ample postoperative contact with other patients (n = 70) or to rooms that were physically located in a manner that afforded little inter-patient contact (n = 9). The results indicate that whereas postoperative depression levels did not differ, patients in the high-patient-contact condition experienced less postoperative anxiety and greater overall satisfaction with their quality of care than did patients in the low-patient-contact condition. Analyses of patients' reported postoperative affiliations suggest several additional benefits of inter-patient contact, such as added emotional support, reduction of uncertainty about what to expect, and the opportunity to compare progress and emotional reactions. The results are consistent with a growing literature that suggests fellow patients can and do serve a useful, adjunctive role in health care. Questions for future research are considered.