Dimeric allostery mechanism of the plant circadian clock photoreceptor ZEITLUPE

In Arabidopsis thaliana, the Light-Oxygen-Voltage (LOV) domain containing protein ZEITLUPE (ZTL) integrates light quality, intensity, and duration into regulation of the circadian clock. Recent structural and biochemical studies of ZTL indicate that the protein diverges from other members of the LOV superfamily in its allosteric mechanism, and that the divergent allosteric mechanism hinges upon conservation of two signaling residues G46 and V48 that alter dynamic motions of a Gln residue implicated in signal transduction in all LOV proteins. Here, we delineate the allosteric mechanism of ZTL via an integrated computational approach that employs atomistic simulations of wild type and allosteric variants of ZTL in the functional dark and light states, together with Markov state and supervised machine learning classification models. This approach has unveiled key factors of the ZTL allosteric mechanisms, and identified specific interactions and residues implicated in functional allosteric changes. The final results reveal atomic level insights into allosteric mechanisms of ZTL function that operate via a non-trivial combination of population-shift and dynamics-driven allosteric pathways.     

CXCR4 expression in prostate cancer progenitor cells

Tumor progenitor cells represent a population of drug-resistant cells that can survive conventional chemotherapy and lead to tumor relapse. However, little is known of the role of tumor progenitors in prostate cancer metastasis. The studies reported herein show that the CXCR4/CXCL12 axis, a key regulator of tumor dissemination, plays a role in the maintenance of prostate cancer stem-like cells. The CXCL4/CXCR12 pathway is activated in the CD44(+)/CD133(+) prostate progenitor population and affects differentiation potential, cell adhesion, clonal growth and tumorigenicity. Furthermore, prostate tumor xenograft studies in mice showed that a combination of the CXCR4 receptor antagonist AMD3100, which targets prostate cancer stem-like cells, and the conventional chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors as compared to monotherapy.     

Stable carbon and nitrogen isotope ratios in muscle and epidermis of arctic whales

Collection of minimally invasive biopsy samples has become an important method to establish normal stable isotopes reference ranges in various wildlife species. Baseline data enhance the understanding of feeding ecology, habitat use, and potential food limitation in apparently healthy, free‐ranging cetaceans. Epidermis and muscle were collected from subsistence‐hunted northern Alaskan bowhead (n= 133 epidermis/134 muscle) and beluga whales (n= 42/49) and subsistence‐hunted Russian gray whales (n= 25/17). Additional samples were obtained from gray whales stranded in California (n= 18/11) during mortality events (1999, 2000). Both δ¹⁵N and δ¹³C are trophic position and benthic/pelagic feeding indicators, respectively, in muscle and epidermis. Epidermis is generally enriched in ¹⁵N over muscle, while epidermal ¹³C is more depleted. Lipid extraction does not alter δ¹⁵N in either tissue, but affects epidermal δ¹³C. Nitrogen‐15 is enriched in muscle, but not epidermis of stranded compared to subsistence‐hunted gray whales, indicating probable protein catabolism and nutritional stress in stranded whales. Similarly, epidermal δ¹³C of harvested whales is lower than in stranded whales, suggesting depleted lipid stores and/or food limitation in stranded animals. Epidermal isotope signatures are similar in both present‐day bowheads and in an ancient sample from the Northern Bering Sea region. Although only one specimen, this suggests trophic level of the ancient whale compares to modern bowheads after a millennium.     

Zinc(II)-mediated inhibition of a ribonuclease by an N-hydroxyurea nucleotide

The inhibition of ribonuclease Bi by 3'-N-hydroxyurea-3'-deoxythymidine 5'-phosphate is enhanced by 30-fold in the presence of Zn(2+). Thus, an N-hydroxyurea nucleotide can recruit Zn(2+) to inhibit the enzymatic activity of a ribonuclease. This result engenders a general strategy for the inhibition of non-metalloenzymes by metal complexes.     

Chronic expression of RCAN1-1L protein induces mitochondrial autophagy and metabolic shift from oxidative phosphorylation to glycolysis in neuronal cells

Expression of the RCAN1 gene can be induced by multiple stresses. RCAN1 proteins (RCAN1s) have both protective and harmful effects and are implicated in common human pathologies. The mechanisms by which RCAN1s function, however, remain poorly understood. We identify RCAN1s as regulators of mitochondrial autophagy (mitophagy) and demonstrate that induction of RCAN1-1L can cause dramatic degradation of mitochondria. The mechanisms of such degradation involve the adenine nucleotide translocator and mitochondrial permeability transition pore opening. We also demonstrate that RCAN1-1L induction can shift cellular bioenergetics from aerobic respiration to glycolysis, yet RCAN1-1L has very little effect on cell division, whereas it has a cumulative negative effect on cell survival. These results shed the light on mechanisms by which RCAN1s can protect or harm cells and by which they may operate in human pathologies. They also suggest that RCAN1s are important players in autophagy and such elusive phenomena as the mitochondrial permeability transition pore.     

The comparison of doses measured by radiochromic films and semiconductor detector in a 175 MeV proton beam

We wanted to verify the response of radiochromic films in a 175 MeV clinical proton beam used at the Joint Institute for Nuclear Research in Dubna against doses measured using semiconductor detectors and compare the results with published data from other centres. Radiochromic films (RCFs) MD-55 and a Vidar VXR-16 scanner were used. The films were irradiated in an unmodulated proton beam and with a beam modulated with a bolus and a ridge filter. Obtained dose distributions were compared with dose distributions measured with a Si-semiconductor detector.For the unmodulated beam the difference between the RCF and the semiconductor detector was 12% in the Bragg peak top. For the modulated beam the difference inside the spread-out Bragg peak region was 4%. Observed deviations between doses measured with RCF and Si-detector outside the Bragg peak were caused by the inhomogeneity of radiochromic emulsion. In the Bragg peak region the RCF doses were lower than those measured by semiconductors. The results were in agreement with published data from other proton therapy centres.     

Norepinephrine induces slow calcium signalling in murine brown preadipocytes through the beta-adrenoceptor/cAMP/protein kinase A pathway

The mechanism of adrenergically activated calcium signalling in isolated murine brown preadipocytes (stromal-vascular fraction) was studied with Fura-2. Norepinephrine (NE) generated in preadipocytes a slow Ca(2+)-response ( approximately 10 nM/min) without a burst and a maximum, whereas in mature brown adipocytes, the quick burst reached 1.5 microM [Ca(2+)](i). Thapsigargin, which is known to discharge Ca(2+) ions from the IP(3)-sensitive stores, initiated a huge capacitative calcium entry in mature brown adipocytes but failed to stimulate a response in preadipocytes. The beta-selective antagonist nadolol almost completely prevented the effect of NE on [Ca(2+)](i), while the antagonist of alpha-adrenoceptors phentolamine caused only a approximately 25% reduction of the cellular response. Forskolin or the cell-permeable Br-cAMP caused [Ca(2+)](i) rise, which were even higher than with NE. The protein kinase A (PKA) inhibitor N-[2-(p-bromocynnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) reduced and the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX), N-cyclohexyl-N-(2-hydroxyethyl)-4-(6-(1,2-dihydro-2-oxoquinolyloxy))butyramide (OPC-3911), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidone (Ro 20-1724) or the protein phosphatase inhibitor okadaic acid enhanced the NE-, isoproterenol- or forskolin-initiated cellular calcium responses. It was concluded that (i) brown preadipocytes lacked a trigger mechanism of initiation of [Ca(2+)](i) rises and (ii) the cAMP- and protein kinase A-mediated phosphorylation played an important role in the beta-adrenoceptor-initiated calcium signalling in these cells. All these features distinguish brown adipocyte precursors from differentiated brown adipocytes, where calcium signalling is initiated exclusively via alpha(1)-adrenoceptors and the trigger mechanism.     

A new subfamily of structurally related human F-box proteins

F-box proteins, a critical component of the evolutionary conserved ubiquitin-protein ligase complex SCF (Skp1/Cdc53-Cullin1/F-box), recruit substrates for ubiquitination and consequent degradation through their specific protein-protein interaction domains. Here, we report the identification of full-length cDNAs encoding three novel human F-box proteins named FBG3, FBG4 and FBG5 which display similarity with previously identified NFB42 (FBX2) and FBG2 (FBX6) proteins. All five proteins are characterized by an approximately 180-amino-acid (aa) conserved C-terminal domain and thus constitute a third subfamily of mammalian F-box proteins. Analysis of genomic organization of the five FBG genes revealed that all of them consist of six exons and five introns. FBG1, FBG2 and FBG3 genes are located in tandem on chromosome 1p36, and FBG4 and FBG5 are mapped to chromosome 19q13. FBG genes are expressed in a limited number of human tissues including kidney, liver, brain and muscle tissues. Expression of rat FBG2 gene was found related to differentiation/proliferation status of hepatocytes. Specifically, FBG2 mRNA was expressed in foetal liver, decreased after birth and re-accumulated in adult liver. Expression of FBG2 was strongly inhibited in hepatoma cells by okadaic acid.