Prevention of mammary tumorigenesis by intermittent caloric restriction: does caloric intake during refeeding modulate the response?

Chronic caloric restriction (CCR) prevents mammary tumorigenesis in rodents, but a protective effect for intermittent caloric restriction (ICR) is less well documented. We recently reported that ICR reduced mammary tumor (MT) incidence of mouse mammary tumor virus-transforming growth factor (MMTV-TGF)-alpha mice to a greater extent than did CCR. Here, we repeated this protocol and obtained serum and tissue samples. Ad libitum (AL) MMTV-TGF-alpha mice were fed AIN-93M diet. Beginning at 10 weeks of age, ICR mice received isocaloric AIN-93M-mod diet (2-fold increases in protein, fat, vitamins, and minerals) at 50% of ad libitum for 3 weeks followed by 3 weeks refeeding with AIN-93M diet. CCR mice were pair-fed AIN-93M:AIN-93M-mod (2:1) matching intakes for restriction/refeeding cycles. Mice were sacrificed for MT size, at 79 (end of 12th restriction) or at 80 (1 week after 12th refeeding) weeks of age. AL and ICR-80 mice had heavier body weights than ICR-79 and CCR mice (P < 0.0001). Cumulative food intakes of ICR and CCR mice were reduced 12% and 15% versus AL mice (P < 0.0001). However, ICR mice consumed significantly (P < 0.0001) more food than did AL mice during refeeding. MT incidence was 84%, 13%, and 27% for AL, ICR, and CCR mice, respectively. MT weight (P < 0.0011) and number (P < 0.01) were higher for AL mice compared with ICR and CCR mice. AL and ICR-80 mice had similar serum IGF-I levels, but only AL values were higher than those of ICR-79 and CCR mice (P < 0.0017). ICR mice had more MT DNA breaks compared with AL and CCR mice, suggesting enhanced apoptosis (P < 0.02). AL mice had higher mammary fat pad ObR and ObRb leptin receptor mRNA expression than did ICR and CCR mice (P < 0.001), but there was no effect on MTs. These results confirm that ICR prevents development of MTs to a greater extent than does CCR, although "overeating" during refeeding may compromise this protection.     

Purification of a soluble casein kinase II from Dictyostelium discoideum lacking the β subunit: regulation during proliferation and differentiation

A type II casein kinase has been purified from the soluble fraction of Dictyostelium discoideum vegetative cells. The enzyme has been purified 370 fold and behaves catalytically as casein kinase type II, in the sense that it utilizes GTP as well as ATP as phosphoryl donors, it is inhibited by low heparin concentrations and phosphorylates a specific peptide for CK II. It is a tetramer of 38 kDa-subunits with catalytic activity and ability to autophosphorylate in vitro. The comparison of this activity with the nuclear enzyme previously purified from the same organism indicates that both have the same molecular structure. Both enzymes have antigenic determinants in common with casein kinase II from bovine thymus, suggesting a high degree of conservation during evolution. Studies on the activity of this enzyme during early differentiation, and in the transition from quiescence to proliferation shows an increase in specific activity suggesting a crucial role for the enzyme in this organism. (Mol Cell Biochem 118: 49–60, 1992)Abbreviations CK Casein Kinase - TLCK N--p Tosyl Lysil-Chloromethylketone - SDS Sodium Dodecyl Sulphate - PAGE Polyacrylamide Gel Electrophoresis - R₃E₃TE₃ Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu - TCA Trichloroacetic Acid     

Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe–S cluster biogenesis regulation

Iron–sulfur (Fe–S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe–S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe–S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe–S cluster biogenesis does not meet cellular demands.     

Plant-specific regulation of replication protein A2 (OsRPA2) from rice during the cell cycle and in response to ultraviolet light exposure

DNA replication is a process that is highly conserved among eukaryotes. Nonetheless, little is known about the proteins involved in it in plants. Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein with several functions in DNA metabolism in humans and yeast and supposedly also in plants. Here we report on the regulation of OsRPA2, the 32-kDa subunit of RPA from rice ( Oryza sativa L.). We found conserved regulation mechanisms at the level of gene expression between animal and plant RPA2 genes and distinct features of OsRPA2 regulation at the level of protein expression. Unlike in animals or in yeast, protein abundance in rice was regulated in a cell cycle phase-specific manner and was altered after UV-C light exposure. On the other hand, posttranslational modification through phosphorylation did not appear to play a pivotal role in rice as it does in animal cells. Our results indicate that plant-specific mechanisms of regulation have evolved for RPA2 within the generally well-conserved process of DNA replication, suggesting specific requirements for regulation of DNA metabolism in plants as compared to other eukaryotes.     

Fate of the (2Fe-2S)(2+) cluster of Escherichia coli biotin synthase during reaction: a Mössbauer characterization

Biotin synthase, the enzyme which catalyzes the last step of the biosynthesis of biotin, contains only (2Fe-2S)(2+) clusters when isolated under aerobic conditions. Previous results showed that reduction by dithionite or photoreduced deazaflavin converts the (2Fe-2S)(2+) to (4Fe-4S)(2+,+). However, until now, no detailed investigation concerning the fate of the (2Fe-2S)(2+) during reduction under assay conditions (NADPH, flavodoxin, flavodoxin reductase) has been realized. Here, we show by M?ssbauer spectroscopy on a partially purified fraction overexpressing the enzyme that, in the presence of a S(2)(-) source and Fe(2+), there is conversion of the predominant (2Fe-2S)(2+) clusters into a 1:1 mixture of (2Fe-2S)(2+) and (4Fe-4S)(2+). No change in this cluster composition was observed in the presence of the physiological reducing system. When the reaction was allowed to proceed by addition of the substrate dethiobiotin, the (4Fe-4S)(2+) was untouched whereas the (2Fe-2S)(2+) was degraded into a new species. This is consistent with the hypothesis that the reduced (4Fe-4S) cluster is involved in mediating the cleavage of AdoMet and that the (2Fe-2S)(2+) is the sulfur source for biotin.     

CCAAT/enhancer binding protein β expression is increased in the brain during HIV-1-infection and contributes to regulation of astrocyte tissue inhibitor of metalloproteinase-1

ABSTRACT: Selective inhibition of GluN2B-containing NMDA receptor (GluN2BR) in spinal dorsal horn effectively alleviates inflammatory pain, suggesting the up-regulation of GluN2BR function involved in central sensitization. Previous studies have demonstrated that the increase in GluN2BR synaptic expression serves as a key step to enhance GluN2BR function after intradermal injection of Complete Freund's Adjuvant (CFA). Here, we showed that cAMP-dependent protein kinase (PKA) played an important role in redistributing GluN2BR at synapses, because inhibition of PKA activity impaired GluN2BR accumulation at post-synaptic density (PSD)-enriched fraction in CFA-injected mice, and direct stimulation of PKA in na?ve mice mimicked the effect of CFA by recruiting GluN2BR at PSD fraction to evoke pain sensitization. Analysis of PKA-initiated signalings unraveled that PKA was able to activate Src-family protein tyrosine kinases member Fyn, possibly by disrupting Fyn association with its inhibitory partner striatal-enriched protein tyrosine phosphatase 61. The active Fyn then promoted GluN2B phosphorylation at Tyr1472, a molecular event known to prevent GluN2BR endocytosis. As a result, pharmacological or genetic manipulation of Fyn activity greatly depressed GluN2BR accumulation at PSD-enriched fraction and ameliorated mechanical allodynia induced by PKA. Our data thus elucidated a critical role of PKA/Fyn/GluN2B signaling in triggering GluN2BR hyperfunction and pain hypersensitivity.     

The β-domain of cluster 2b streptokinase is a major determinant for the regulation of its plasminogen activation activity by cellular plasminogen receptors

Cluster 2b streptokinase (SK2b), secreted by invasive skin-trophic strains of Streptococcus pyogenes (GAS), is a human plasminogen (hPg) activator that optimally functions when human plasma hPg is bound, via its kringle-2 domain, to cognizant bacterial cells through the a1a2 domain of the major cellular hPg receptor, Plasminogen-binding group A streptococcal M-like protein (PAM). Another class of streptokinases (SK1), secreted primarily by GAS strains that possess affinity for pharyngeal infections, does not require PAM-bound hPg for optimal activity. We find herein that replacement of the central β-domain of SK2b with the same module from SK1 reduces the dependency of SK2b on PAM, and the converse is true when the β-domain of SK1 is replaced with this same region of SK2b. These data suggest that simple evolutionary shuttling of protein domains in GAS can be employed by GAS to rapidly generate strains that differ in tissue tropism and invasive capability and allow the bacteria to survive different challenges by the host.     

Comprehensive survey of the VxGΦL motif of PP2Cs from Oryza sativa reveals the critical role of the fourth position in regulation of ABA responsiveness

Plant Molecular Biology - Regulation of abscisic acid (ABA) signaling is crucial in balancing responses to abiotic stresses and retaining growth in planta. An ABA receptor (PYL/RCAR) and a protein...     

Novel insight into the function and regulation of αN-catenin by Snail2 during chick neural crest cell migration

The neural crest is a transient population of migratory cells that differentiates to form a variety of cell types in the vertebrate embryo, including melanocytes, the craniofacial skeleton, and portions of the peripheral nervous system. These cells initially exist as adherent epithelial cells in the dorsal aspect of the neural tube and only later become migratory after an epithelial-to-mesenchymal transition (EMT). Snail2 plays a critical role in mediating chick neural crest cell EMT and migration due to its expression by both premigratory and migratory cranial neural crest cells and its ability to down-regulate intercellular junctions components. In an attempt to delineate the role of cellular junction components in the neural crest, we have identified the adherens junction molecule neural alpha-catenin (αN-catenin) as a Snail2 target gene whose repression is critical for chick neural crest cell migration. Knock-down and overexpression of αN-catenin enhances and inhibits neural crest cell migration, respectively. Furthermore, our results reveal that αN-catenin regulates the appropriate movement of neural crest cells away from the neural tube into the embryo. Collectively, our data point to a novel function of an adherens junction protein in facilitating the proper migration of neural crest cells during the development of the vertebrate embryo.     

Is a Ca2+ -ATPase involved in Ca2+ regulation during capacitation and the acrosome reaction of guinea-pig spermatozoa?

The Ca2+-ATPase antagonists quercetin and ethacrynic acid accelerated the onset of the acrosome reaction in guinea-pig spermatozoa incubated in the continuous presence of Ca2+, whereas furosemide had no effect, and sodium orthovanadate only affected sperm motility. When spermatozoa were preincubated in a 'Ca2+-free' medium, quercetin and ethacrynic acid shortened capacitation time: spermatozoa incubated for 1 h in 100-200 microM-ethacrynic acid showed 60-80% acrosome reactions when Ca2+ was added. Such spermatozoa were able to fertilize zona-free hamster eggs. Our results therefore point to the possible involvement of a Ca2+-ATPase in the regulation of intracellular Ca2+ in spermatozoa. Cysteine and dithiothreitol, both disulphide reducing agents, prevented the effects of quercetin and ethacrynic acid, suggesting that sulphydryl groups may be important for the expression of Ca2+-ATPase activity. Lysophosphatidylserine (LS) also prevented the stimulatory effect of ethacrynic acid, an effect similar to that shown by LS on lysophosphatidylcholine (LC). It is argued that both LS and LC could exert their action through an effect on the Ca2+-ATPase.