PKR activity is required for acute leukemic cell maintenance and growth: A role for PKR‐mediated phosphatase activity to regulate GSK‐3 phosphorylation

Recent reports demonstrate that PKR is constitutively active in a variety of tumors and is required for tumor maintenance and growth. Here we report acute leukemia cell lines contain elevated levels of p‐T451 PKR and PKR activity as compared to normal controls. Inhibition of PKR with a specific inhibitor, as well as overexpression of a dominant‐negative PKR, inhibited cell proliferation and induced cell death. Interestingly, PKR inhibition using the specific inhibitor resulted in a time‐dependent augmentation of AKT S473 and GSK‐3α S21 phosphorylation, which was confirmed in patient samples. Increased phosphorylation of AKT and GSK‐3α was not dependent on PI3K activity. PKR inhibition augmented levels of p‐S473 AKT and p‐S21/9 GSK‐3α/β in the presence of the PI3K inhibitor, LY294002, but was unable to augment GSK‐3α or β phosphorylation in the presence of the AKT inhibitor, A443654. Pre‐treatment with the PKR inhibitor blocked the ability of A443654 and LY294002 to promote phosphorylation of eIF2α, indicating the mechanism leading to AKT phosphorylation and activation did not require eIF2α phosphorylation. The effects of PKR inhibition on AKT and GSK‐3 phosphorylation were found to be, in part, PP2A‐dependent. These data indicate that, in acute leukemia cell lines, constitutive basal activity of PKR is required for leukemic cell homeostasis and growth and functions as a negative regulator of AKT, thereby increasing the pool of potentially active GSK‐3. J. Cell. Physiol. 221: 232–241, 2009. © 2009 Wiley‐Liss, Inc     

Sensitivity of methods for estimating breeding values using genetic markers to the number of QTL and distribution of QTL variance

The objective of this simulation study was to compare the effect of the number of QTL and distribution of QTL variance on the accuracy of breeding values estimated with genomewide markers (MEBV). Three distinct methods were used to calculate MEBV: a Bayesian Method (BM), Least Angle Regression (LARS) and Partial Least Square Regression (PLSR). The accuracy of MEBV calculated with BM and LARS decreased when the number of simulated QTL increased. The accuracy decreased more when QTL had different variance values than when all QTL had an equal variance. The accuracy of MEBV calculated with PLSR was affected neither by the number of QTL nor by the distribution of QTL variance. Additional simulations and analyses showed that these conclusions were not affected by the number of individuals in the training population, by the number of markers and by the heritability of the trait. Results of this study show that the effect of the number of QTL and distribution of QTL variance on the accuracy of MEBV depends on the method that is used to calculate MEBV.     

Plasma factors controlling atrial natriuretic peptide (ANP) aggregation: role of lipoproteins

We have previously shown that human plasma atrial alpha-natriuretic peptide (alpha-hANP) sequestering is a protective phenomenon against amyloid aggregation. In the present work, the possible role of lipoproteins as alpha-hANP binding factors has been investigated in vitro using an experimental model, developed in our laboratory, that allows to work at physiological concentrations. This approach consists of gel filtration on Sephacryl S-300 HR of big alpha-[(125)I]hANP generated in phosphate buffered saline or in human normal plasma supplemented or not with lipoproteins. The results of these experiments indicate that high density lipoproteins (HDL) are responsible for the ANP binding phenomenon observed in vitro, while low density lipoproteins and very low density lipoproteins do not directly interact with ANP. Moreover, the HDL remodeling process occurring in vitro has been analyzed during plasma incubation by monitoring the redistribution of lipids and apolipoproteins among the HDL subclasses. The changes in HDL size and composition observed in incubated plasma were compared with the redistribution of endogenous and labeled big ANP. The obtained results revealed that both tend to follow the molecular rearrangement in plasma of apolipoprotein A-I containing particles and suggested that, among HDL species, the small particles are mainly involved in the ANP binding phenomenon. This hypothesis was further demonstrated by ligand blotting experiments that confirmed the existence of differences in the ability of HDL particles to bind alpha-[(125)I]hANP.     

Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle

Proliferation of mammalian cardiomyocytes stops rapidly after birth and injured hearts do not regenerate adequately. High cyclin-dependent kinase inhibitor (CKI) levels have been observed in cardiomyocytes, but their role in maintaining cardiomyocytes in a post-mitotic state is still unknown. In this report, it was investigated whether CKI knockdown by RNA interference induced cardiomyocyte proliferation. We found that triple transfection with p21(Waf1), p27(Kip1), and p57(Kip2) siRNAs induced both neonatal and adult cardiomyocyte to enter S phase and increased the nuclei/cardiomyocyte ratio; furthermore, a subpopulation of cardiomyocytes progressed beyond karyokynesis, as assessed by the detection of mid-body structures and by straight cardiomyocyte counting. Intriguingly, cardiomyocyte proliferation occurred in the absence of overt DNA damage and aberrant mitotic figures. Finally, CKI knockdown and DNA synthesis reactivation correlated with a dramatic change in adult cardiomyocyte morphology that may be a prerequisite for cell division. In conclusion, CKI expression plays an active role in maintaining cardiomyocyte withdrawal from the cell cycle.     

Vertebrae numbers of the early hominid lumbar spine

General doctrine holds that early hominids possessed a long lumbar spine with six segments. This is mainly based on Robinson's (1972) interpretation of a single partial Australopithecus africanus skeleton, Sts 14, from Sterkfontein, South Africa. As its sixth last presacral vertebra exhibits both thoracic and lumbar characteristics, current definitions of lumbar vertebrae and lumbar ribs are discussed in the present study. A re-analysis of its entire preserved vertebral column and comparison with Stw 431, another partial A. africanus skeleton from Sterkfontein, and the Homo erectus skeleton KNM-WT 15000 from Nariokotome, Kenya, did not provide strong evidence for the presence of six lumbar vertebrae in either of these early hominids. Thus, in Sts 14 the sixth last presacral vertebra has on one side a movable rib. In Stw 431, the corresponding vertebra shows indications for a rib facet. In KNM-WT, 15000 the same element is very fragmentary, but the neighbouring vertebrae do not support the view that it is L1. Although in all three fossils the transitional vertebra at which the articular facets change orientation seems to be at Th11, this is equal to a large percentage of modern humans. Indeed, a modal number of five lumbar vertebrae, as in modern humans, is more compatible with evolutionary principles. For example, six lumbar vertebrae would require repetitive shortening and lengthening not only of the lumbar, but also of the entire precaudal spine. Furthermore, six lumbar vertebrae are claimed to be biomechanically advantageous for early hominid bipedalism, yet an explanation is lacking as to why the lumbar region should have shortened in later humans. All this raises doubts about previous conclusions for the presence of six lumbar vertebrae in early hominids. The most parsimonious explanation is that they did not differ from modern humans in the segmentation of the vertebral column.     

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non‐native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non‐native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large‐scale hybridization and introgression.     

On the association of DNA polymerase alpha activity with the nuclear matrix in HeLa cells

The association of DNA polymerase alpha activity with the nuclear matrix has been reinvestigated in HeLa cells. Isolated nuclei were extracted with 2M NaCl and then digested with Dnase I and the final structures were recovered by centrifugation through a sucrose cushion. Typically over 98% of the total DNA synthesized in the matrix fraction on either endogenous matrix-associated DNA or activated calf thymus DNA was due to DNA polymerase alpha as defined by inhibition to n-ethylmaleimide or aphidicolin. DNA polymerase beta activity was absent or recovered in only trace amounts. Matrix-bound DNA polymerase alpha activity demonstrated a remarkable degree of stability: DNA synthesis was essentially linear up to 3 hours at 37 degrees C. Overall, these results substantiate previous findings from regenerating rat liver, unlike other data obtained from tissue culture cells.     

Enhanced Slow-Wave EEG Activity and Thermoregulatory Impairment following the Inhibition of the Lateral Hypothalamus in the Rat

Neurons within the lateral hypothalamus (LH) are thought to be able to evoke behavioural responses that are coordinated with an adequate level of autonomic activity. Recently, the acute pharmacological inhibition of LH has been shown to depress wakefulness and promote NREM sleep, while suppressing REM sleep. These effects have been suggested to be the consequence of the inhibition of specific neuronal populations within the LH, i.e. the orexin and the MCH neurons, respectively. However, the interpretation of these results is limited by the lack of quantitative analysis of the electroencephalographic (EEG) activity that is critical for the assessment of NREM sleep quality and the presence of aborted NREM-to-REM sleep transitions. Furthermore, the lack of evaluation of the autonomic and thermoregulatory effects of the treatment does not exclude the possibility that the wake-sleep changes are merely the consequence of the autonomic, in particular thermoregulatory, changes that may follow the inhibition of LH neurons. In the present study, the EEG and autonomic/thermoregulatory effects of a prolonged LH inhibition provoked by the repeated local delivery of the GABA_A agonist muscimol were studied in rats kept at thermoneutral (24°C) and at a low (10°C) ambient temperature (Ta), a condition which is known to depress sleep occurrence. Here we show that: 1) at both Tas, LH inhibition promoted a peculiar and sustained bout of NREM sleep characterized by an enhancement of slow-wave activity with no NREM-to-REM sleep transitions; 2) LH inhibition caused a marked transitory decrease in brain temperature at Ta 10°C, but not at Ta 24°C, suggesting that sleep changes induced by LH inhibition at thermoneutrality are not caused by a thermoregulatory impairment. These changes are far different from those observed after the short-term selective inhibition of either orexin or MCH neurons, suggesting that other LH neurons are involved in sleep-wake modulation.     

Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.     

Intramembrane client recognition potentiates the chaperone functions of calnexin

One‐third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar‐binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin‐based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.