Comparison of properties of tests for assessing tumor clonality

SUMMARY: In a recent article Begg et al. (2007, Biometrics 63, 522-530) proposed a statistical test to determine whether or not a diagnosed second primary tumor is biologically independent of the original primary tumor, by comparing patterns of allelic losses at candidate genetic loci. The proposed concordant mutations test is a conditional test, an adaptation of Fisher's exact test, that requires no knowledge of the marginal mutation probabilities. The test was shown to have generally good properties, but is susceptible to anticonservative bias if there is wide variation in mutation probabilities between loci, or if the individual mutation probabilities of the parental alleles for individual patients differ substantially from each other. In this article, a likelihood ratio test is derived in an effort to address these validity issues. This test requires prespecification of the marginal mutation probabilities at each locus, parameters for which some information will typically be available in the literature. In simulations this test is shown to be valid, but to be considerably less efficient than the concordant mutations test for sample sizes (numbers of informative loci) typical of this problem. Much of the efficiency deficit can be recovered, however, by restricting the allelic imbalance parameter estimate to a prespecified range, assuming that this parameter is in the prespecified range.     

Helicobacter pylori CagA-dependent macrophage migration inhibitory factor produced by gastric epithelial cells binds to CD74 and stimulates procarcinogenic events

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has recently been implicated in carcinogenesis. Helicobacter pylori, which is closely linked to gastric cancer, induces the gastric epithelium to produce proinflammatory cytokines, including MIF. MIF can bind to CD74, which we have previously shown to be highly expressed on the surface of gastric epithelial cells (GEC) during H. pylori infection. In this study, we sought to investigate the role of the H. pylori-induced MIF on epithelial proliferation and procarcinogenic events. Upon establishing a role for the H. pylori CagA virulence factor in MIF production, MIF binding to CD74 on GEC was confirmed. rMIF and H. pylori were shown to increase GEC proliferation, which was decreased when cagA- strains were used and when CD74 was blocked by mAbs. Apoptosis was also decreased by MIF, but increased by cagA- strains that induced much lower amounts of MIF than the wild-type bacteria. Furthermore, MIF binding to CD74 was also shown to decrease p53 phosphorylation and up-regulate Bcl-2 expression. This data describes a novel system in which an H. pylori virulence factor contributes to the production of a host factor that in turn up-regulates procarcinogenic events by the gastric epithelium.     

Fluorescence changes accompanying short-term light adaptations in photosystem I and photosystem II of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 and phycobiliprotein-impaired mutants: State 1/State 2 transitions and carotenoid-induced quenching of phycobilisomes

The features of the two types of short-term light-adaptations of photosynthetic apparatus, State 1/State 2 transitions, and non-photochemical fluorescence quenching of phycobilisomes (PBS) by orange carotene-protein (OCP) were compared in the cyanobacterium Synechocystis sp. PCC 6803 wild type, CK pigment mutant lacking phycocyanin, and PAL mutant totally devoid of phycobiliproteins. The permanent presence of PBS-specific peaks in the in situ action spectra of photosystem I (PSI) and photosystem II (PSII), as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 690 nm (PSII) and 725 nm (PSI) showed that PBS are constitutive antenna complexes of both photosystems. The mutant strains compensated the lack of phycobiliproteins by higher PSII content and by intensification of photosynthetic linear electron transfer. The detectable changes of energy migration from PBS to the PSI and PSII in the Synechocystis wild type and the CK mutant in State 1 and State 2 according to the fluorescence excitation spectra measurements were not registered. The constant level of fluorescence emission of PSI during State 1/State 2 transitions and simultaneous increase of chlorophyll fluorescence emission of PSII in State 1 in Synechocystis PAL mutant allowed to propose that spillover is an unlikely mechanism of state transitions. Blue–green light absorbed by OCP diminished the rout of energy from PBS to PSI while energy migration from PBS to PSII was less influenced. Therefore, the main role of OCP-induced quenching of PBS is the limitation of PSI activity and cyclic electron transport under relatively high light conditions.     

Lipoprotein-associated phospholipase A2 activity in patients with preserved left ventricular ejection fraction

Background: A significant proportion of heart failure (HF) patients have preserved ejection fraction (EF). Considering that inflammation and oxidative stress are involved in HF evolution, we investigated lipoprotein-associated phospholipase A2 (LpPLA2), an enzyme involved in these pathophysiologic processes in relation to EF. Methods and results: The study included 208 HF patients and 20 healthy controls. HF patients with preserved EF (HFpEF) represented 42.31% of all HF patients. LpPLA2 activity was significantly increased in HF patients when compared with controls and was higher in HFpEF than in HF with reduced EF patients (HFrEF). The incidence of left ventricular hypertrophy was higher in HFpEF than in HFrEF (EF < 50). Conclusion: Confirming its role as a marker of vascular inflammation, LpPLA2 seems to be a biomarker constantly correlated with HF, regardless of etiology. Elevated plasma values of LpPLA2 in HFpEF are consistent with the exacerbated inflammatory status.     

eIF2-dependent and eIF2-independent modes of initiation on the CSFV IRES: a common role of domain II

Specific interactions of the classical swine fever virus internal ribosomal entry site (IRES) with 40S ribosomal subunits and eukaryotic translation initiation factor (eIF)3 enable 43S preinitiation complexes containing eIF3 and eIF2-GTP-Met-tRNA(iMet) to bind directly to the initiation codon, yielding 48S initiation complexes. We report that eIF5B or eIF5B/eIF3 also promote Met-tRNA(iMet) binding to IRES-40S complexes, forming 48S complexes that can assemble elongation-competent ribosomes. Although 48S complexes assembled both by eIF2/eIF3- and eIF5B/eIF3-mediated Met-tRNA(iMet) recruitment were destabilized by eIF1, dissociation of 48S complexes formed with eIF2 could be out-competed by efficient subunit joining. Deletion of IRES domain II, which is responsible for conformational changes induced in 40S subunits by IRES binding, eliminated the sensitivity of 48S complexes assembled by eIF2/eIF3- and eIF5B/eIF3-mediated mechanisms to eIF1-induced destabilization. However, 48S complexes formed by the eIF5B/eIF3-mediated mechanism on the truncated IRES could not undergo efficient subunit joining, as reported previously for analogous complexes assembled with eIF2, indicating that domain II is essential for general conformational changes in 48S complexes, irrespective of how they were assembled, that are required for eIF5-induced hydrolysis of eIF2-bound GTP and/or subunit joining.     

Kinetics of actin unfolding induced by guanidine hydrochloride.

The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy.     

Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment

Background

Dehydrins are known as Group II late embryogenesis abundant proteins. Their high hydrophilicity and thermostability suggest that they may be structure stabilizers with detergent and chaperone-like properties. They are localised in the nucleus, cytoplasm, and plasma membrane. We have recently found putative dehydrins in the mitochondria of some cereals in response to cold. It is not known whether dehydrin-like proteins accumulate in plant mitochondria in response to stimuli other than cold stress.

Results

We have found five putative dehydrins in the mitochondria of winter wheat, rye and maize seedlings. Two of these polypeptides had the same molecular masses in all three species (63 and 52 kD) and were thermostable. Drought, freezing, cold, and exogenous ABA treatment led to higher accumulation of dehydrin-like protein (dlp) 63 kD in the rye and wheat mitochondria. Protein 52 kD was induced by cold adaptation and ABA. Some accumulation of these proteins in the maize mitochondria was found after cold exposition only. The other three proteins appeared to be heat-sensitive and were either slightly induced or not induced at all by all treatments used.

Conclusions

We have found that, not only cold, but also drought, freezing and exogenous ABA treatment result in accumulation of the thermostable dehydrins in plant mitochondria. Most cryotolerant species such as wheat and rye accumulate more heat-stable dehydrins than cryosensitive species such as maize. It has been supposed that their function is to stabilize proteins in the membrane or in the matrix. Heat-sensitive putative dehydrins probably are not involved in the stress reaction and adaptation of plants.     

Host S-nitrosylation inhibits clostridial small molecule-activated glucosylating toxins

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins. Virulence is dependent on the autoactivation of a toxin cysteine protease, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP(6)). Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP(6)- and inositol pyrophosphate (InsP(7))-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP(6) enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.     

Integrated analysis of the conformation of a protein-linked spin label by crystallography,EPR and NMR spectroscopy

Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein–protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.