The ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory group

Background

Protein homeostasis in the endoplasmic reticulum (ER) has recently emerged as a therapeutic target for cancer treatment. Disruption of ER homeostasis results in ER stress, which is a major cause of cell death in cells exposed to the proteasome inhibitor Bortezomib, an anti-cancer drug approved for treatment of multiple myeloma and Mantle cell lymphoma. We recently reported that the ERAD inhibitor Eeyarestatin I (EerI) also disturbs ER homeostasis and has anti-cancer activities resembling that of Bortezomib.

Methodology and Principal Findings

Here we developed in vitro binding and cell-based functional assays to demonstrate that a nitrofuran-containing (NFC) group in EerI is the functional domain responsible for the cytotoxicity. Using both SPR and pull down assays, we show that EerI directly binds the p97 ATPase, an essential component of the ERAD machinery, via the NFC domain. An aromatic domain in EerI, although not required for p97 interaction, can localize EerI to the ER membrane, which improves its target specificity. Substitution of the aromatic module with another benzene-containing domain that maintains membrane localization generates a structurally distinct compound that nonetheless has similar biologic activities as EerI.

Conclusions and Significance

Our findings reveal a class of bifunctional chemical agents that can preferentially inhibit membrane-bound p97 to disrupt ER homeostasis and to induce tumor cell death. These results also suggest that the AAA ATPase p97 may be a potential drug target for cancer therapeutics.     

Effect of pituitary grafting on ovarian oestradiol secretion in the hypophysectomized chick embryo.

Chick embryos were hypophysectomized by partial decapitation at the stage of 42 h of incubation and grafted with a hypophysis from a 12-days-old donor embryo on the chorio-allantoic membrane at 9 1/2 days. Two days later, their ovary was removed for organ culture and its oestradiol secretion rate was compared to that of the ovary of hypophysectomized, non grafted control embryos. The oestradiol secretion rate in the grafted embryos was almost twice that in the hypophysioprivic embryos and in the range of that in normal embryos. This result suggests that the hypophysis controls oestradiol secretion of the ovary in the 11 1/2-days-old chick embryo.     

CLL cells are resistant to smac mimetics because of an inability to form a ripoptosome complex

In the lymph node (LN) environment, chronic lymphocytic leukemia (CLL) cells display increased NF-κB activity compared with peripheral blood CLL cells, which contributes to chemoresistance. Antagonists of cellular inhibitor of apoptosis proteins (cIAPs) can induce apoptosis in various cancer cells in a tumor necrosis factor-α (TNFα)-dependent manner and are in preclinical development. Smac-mimetics promote degradation of cIAP1 and cIAP2, which results in TNFR-mediated apoptosis via formation of a ripoptosome complex, comprising RIPK1, Fas-associated protein with death domain, FLICE-like inhibitory protein and caspase-8. CD40 stimulation of CLL cells in vitro is used as a model to mimic the LN microenvironment and results in NF-κB activation and TNFα production. In this study, we investigated the response of CLL cells to smac-mimetics in the context of CD40 stimulation. We found that treatment with smac-mimetics results in cIAP1 and cIAP2 degradation, yet although TNFα is produced, this did not induce apoptosis. Despite the presence of all components, the ripoptosome complex did not form upon smac-mimetic treatment in CLL cells. Thus, CLL cells seem to possess aberrant upstream NF-κB regulation that prevents ripoptosome formation upon IAP degradation. Unraveling the exact molecular mechanisms of disturbed ripoptosome formation may offer novel targets for treatment in CLL.     

Effect of cAMP and FSH on the production of estrogens by rat fetal gonads cultured in vitro

The regulation of aromatase activity by cAMP and FSH has been demonstrated in the prepubertal rat testis and ovary, and the question posed whether this regulation was already operative at foetal stages. In the present study, testes and ovaries from 17- to 21-day-old rat foetuses were cultured in vitro in the presence of [3H]-testosterone and in the presence or absence of cAMP or FSH. Oestrone and oestradiol formed from [3H]-testosterone were measured by double isotopic dilution and recrystallization to constant specific activity. Aromatase activity was augmented in both gonads by cAMP, but only in the testis by FSH. Thus, the regulation of aromatase activity by FSH begins earlier in the testis than in the ovary.     

Die wirkung von hyperthermie auf DNA-reparaturvorgänge

Summary The time-course of DNA repair after irradiation was measured in HeLa cells at various temperatures. Unscheduled DNA synthesis was estimated by incorporation of³H-thymidine in presence of hydroxyurea. To dedect the ligase reaction, the number of single strand breaks (SSB) was determined by centrifugation in alcaline sucrose as well as by hydroxylapatite chromatography after partial denaturation. In addition, the temperature dependence of DNA polymerase and DNase reaction in cell-free systems were measured. These data were compared with the reduction of colony-forming ability of the cells caused by irradiation and following repair at various temperatures. All steps of repair proceed faster at 41–43° than at 37° but cells are most resistant to irradiation at 37°. We therefore assume that the DNA repair process at 42° is faster but more error prone than at 37°.

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Die wirkung von hyperthermie auf DNA-reparaturvorgänge

Zusammenfassung In HeLa Zellen wurde der zeitliche Ablauf des DNA Reparaturvorganges nach-Bestrahlung bei verschiedenen Temperaturen gemessen. Die unprogrammierte DNA-Synthese wurde durch Einbau von³H-Thymidin in Gegenwart von Hydroxyharnstoff bestimmt. Für die Erfassung des Ligase Schrittes wurde die Anzahl der DNA-Einstrangbrüche (SSB) durch Zentrifugation im alkalischen Saccharosegradienten sowie durch Hydroxylapatit-Chromatographie nach partieller Denaturierung bestimmt. Weiters wurde die Temperaturabhängigkeit der DNA-Polymerase- und der DNase-Reaktion im zellfreien System gemessen. Diese Daten wurden mit der Verminderung der Fähigkeit zur Kolonie-Bildung der Zellen durch-Bestrahlung und anschließender Reparatur bei verschiedenen Temperaturen verglichen. Es zeigte sich, daß alle Schritte des Reparaturvorganges bei 41–43° schneller ablaufen als bei 37°, die Zellen jedoch bei 37° die größte Strahlenresistenz zeigen. Wir nehmen daher an, daß der Reparaturvorgang bei 42° zwar schneller aber ungenauer abläuft.

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Eingegangen am 7. Dezember 1978     

Phylogenetic and coalescent analysis of three loci suggest that the Water Rail is divisible into two species, <Emphasis Type=Italic>Rallus aquaticus</Emphasis> and <Emphasis Type=Italic>R. indicus</Emphasis>

Background  

Water Rails (Rallus aquaticus) inhabit fragmented freshwater wetlands across their Palearctic distribution. Disjunct populations are now thought to be morphologically similar over their vast geographic range, though four subspecies had been recognized previously. The fossil record suggests that Water Rails (R. aquaticus) were already spread across the Palearctic by the Pleistocene ~2 million years ago, and the oldest fossil remains thought to be closely related to the common ancestor of water rails date from the Pliocene.     

Different models of genetic variation and their effect on genomic evaluation

Background

The theory of genomic selection is based on the prediction of the effects of quantitative trait loci (QTL) in linkage disequilibrium (LD) with markers. However, there is increasing evidence that genomic selection also relies on "relationships" between individuals to accurately predict genetic values. Therefore, a better understanding of what genomic selection actually predicts is relevant so that appropriate methods of analysis are used in genomic evaluations.

Methods

Simulation was used to compare the performance of estimates of breeding values based on pedigree relationships (Best Linear Unbiased Prediction, BLUP), genomic relationships (gBLUP), and based on a Bayesian variable selection model (Bayes B) to estimate breeding values under a range of different underlying models of genetic variation. The effects of different marker densities and varying animal relationships were also examined.

Results

This study shows that genomic selection methods can predict a proportion of the additive genetic value when genetic variation is controlled by common quantitative trait loci (QTL model), rare loci (rare variant model), all loci (infinitesimal model) and a random association (a polygenic model). The Bayes B method was able to estimate breeding values more accurately than gBLUP under the QTL and rare variant models, for the alternative marker densities and reference populations. The Bayes B and gBLUP methods had similar accuracies under the infinitesimal model.

Conclusions

Our results suggest that Bayes B is superior to gBLUP to estimate breeding values from genomic data. The underlying model of genetic variation greatly affects the predictive ability of genomic selection methods, and the superiority of Bayes B over gBLUP is highly dependent on the presence of large QTL effects. The use of SNP sequence data will outperform the less dense marker panels. However, the size and distribution of QTL effects and the size of reference populations still greatly influence the effectiveness of using sequence data for genomic prediction.     

Failure to Use Cubicles and Concentrate Dispenser by Heifers after Transfer from Rearing Accommodation to Milking Herd

Thirty-three dairy farms in the Norwegian counties of ?stfold and Akershus in which cubicle sheds had been in use for at least one year and with a herd size of less than 60 cows, were contacted and asked to participate in a study. The study focused on heifers' use of cubicles and concentrate dispenser just after being transferred from rearing accommodation to the milking herd. For each heifer, the farmer recorded cubicle use once nightly between 9 and 11 pm. The daily amount of concentrate released in the dispenser and the allotted daily ration were also recorded. The recording period was 15 consecutive days for cubicle use and 7 days for concentrate dispenser use. Cubicle refusal behaviour, i.e. lying outside the cubicles, was analysed by logistic regression using rearing accommodation of heifers, herd size, heifer age, and housing layout as independent variables, and herd as a clustering variable. On Day 2 after transfer, 34% of the heifers were showing cubicle refusal behaviour (N = 340). By Day 15 this percentage had dropped to 23. Cubicle refusal was lower throughout the whole period among heifers which used the cubicles on the 3 first days after transfer compared to those which did not. This tendency could also be detected several months later. The analysis showed cubicle refusal to be significantly associated with rearing accommodation (OR = 6.1, c.i._95%OR = 1.5–24.3, P = 0.01) and cubicle layout in the shed (OR = 0.2, c.i._95%OR = 0.0–0.7, P = 0.01). None of the tested variables were found to be significant for failure to use the concentrate dispenser, a behaviour which was less frequent than cubicle refusal. However, 8 percent of the heifers did not visit the dispenser at all throughout the 7 days of observation.     

Single-molecule anisotropy imaging

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D(rot) = 7 x 10(7) rad(2)/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D(lat) = 3.5 x 10(-8) cm(2)/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L(beta) gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D(rot) = 1.2 rad(2)/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.     

Estrogen production by fetal rat gonads

Aromatase activity in fetal rat testes and ovaries was demonstrated by the conversion of tritiated testosterone or 19-hydroxyandrostenedione into estrone and estradiol, which were identified and quantified by double isotopic dilution and recrystallization to constant specific activity. Testes formed mostly estradiol, ovaries mostly estrone. Aromatase activity was stimulated by cAMP in both the testes and ovaries as early as 17 days of fetal life. Stimulation by FSH was noted at this same stage in the testis, but not before 3–4 days after birth in the ovary. LH was without effect on aromatase activity in both kinds of gonads. Basal estrogen secretion was non-existent or undetectable in both the testes and ovaries in fetal stages. In the presence of cAMP and as early as 17 days of fetal life, the testes released estradiol, as early as 14 days the ovaries released estrone. Estrogen secretion was stimulated by LH and FSH at fetal stages in the testis and at infantile stages in the ovary. Responsiveness to gonadotrophins closely followed the appearance of the receptors.