An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication

We have analyzed how single-strand DNA gaps affect DNA replication in Xenopus egg extracts. DNA lesions generated by etoposide, a DNA topoisomerase II inhibitor, or by exonuclease treatment activate a DNA damage checkpoint that blocks initiation of plasmid and chromosomal DNA replication. The checkpoint is abrogated by caffeine and requires ATR, but not ATM, protein kinase. The block to DNA synthesis is due to inhibition of Cdc7/Dbf4 protein kinase activity and the subsequent failure of Cdc45 to bind to chromatin. The checkpoint does not require pre-RC assembly but requires loading of the single-strand binding protein, RPA, on chromatin. This is the biochemical demonstration of a DNA damage checkpoint that targets Cdc7/Dbf4 protein kinase.     

The Escherichia coli DjlA and CbpA proteins can substitute for DnaJ in DnaK-mediated protein disaggregation

The DnaJ (Hsp40) protein of Escherichia coli serves as a cochaperone of DnaK (Hsp70), whose activity is involved in protein folding, protein targeting for degradation, and rescue of proteins from aggregates. Two other E. coli proteins, CbpA and DjlA, which exhibit homology with DnaJ, are known to interact with DnaK and to stimulate its chaperone activity. Although it has been shown that in dnaJ mutants both CbpA and DjlA are essential for growth at temperatures above 37 degrees C, their in vivo role is poorly understood. Here we show that in a dnaJ mutant both CbpA and DjlA are required for efficient protein dissaggregation at 42 degrees C.     

Reversible PEGylation of peptide YY3-36 prolongs its inhibition of food intake in mice

Administration of peptide YY(3-36) (PYY(3-36)) to fasting humans or mice shortly before re-feeding effectively reduced their food intake, but PYY(3-36) exhibited a functional half-life of only approximately 3 h. Attachment of poly(ethylene glycol) to proteins and peptides (PEGylation) prolongs their half-life in vivo, but completely inactivated PYY(3-36). We developed a reversibly PEGylated PYY(3-36) derivative by coupling it to a 40 kDa PEG through a spontaneously cleavable linker. The resulting conjugate (PEG(40)-FMS-PYY(3-36)) gradually released unmodified PYY(3-36) in vivo, exhibiting an eightfold increase in its functional half-life, to approximately 24h. This long-acting PYY(3-36) pro-drug may serve as an effective means for controlling food intake in humans.     

Implication of proteases in the respiration dependent inactivation of the lactose permease of E. coli

The lactose permease of E. coli becomes irreversibly inactivated during lactose transport under conditions of high respiratory activity. This inactivation is characterized by a decrease in the steady state of lactose accumulation, a decrease in the influx rate of lactose, and a decrease in the transmembrane electrical potential. We report here that inhibitors of serine proteases (phenylmethylsulfonyl fluoride and N-alpha-P-tosyl-L-lysine chloromethyl ketone) prevent this inactivation, thus implicating proteases in this process.     

Cyanine dye as monitor of membrane potentials in Escherichia coli cells and membrane vesicles.

The fluorescence response of a positively charged cyanine dye: 3,3'-dimethylindodicarbocyanine iodide can be specifically related to the generation in Escherichia coli cells and E. coli membrane vesicles of an electrical membrane potential induced either by substrate oxidation or by an artificially imposed potassium diffusion gradient. The energy-dependent quenching of the dye fluorescence correlates well with the known effect on delta phi of: oxidation of various energy sources, external pH and solute accumulation. Thus, in the vesicles, the fluorescence quenching of the dye increases from succinate to D-lactate, to ascorbate/phenazine methosulfate and parallels the increasing ability of these electron donors to generate a delta phi. In the vesicles, delta phi is only weakly dependent on external pH, whereas in the cells, delta phi increases with increasing external pH. Lactose accumulation in the vesicles results in the partial utilization of delta phi. A calibration of the dye fluorescence in terms of delta phi has been determined using valinomycin-induced potassium diffusion potential.     

Comparison of lactose uptake in resting and energized Escherichia coli cells: high rates of respiration inactivate the lac carrier

The transport of lactose by Escherichia coli cells was radically different in the absence and in the presence of an exogenous energy source: in the former case, the time course of lactose accumulation was monotonous; in the latter case, lactose accumulation reached a maximum and then decreased to a final steady-state level lower than that observed in the absence of an energy source. We show that this "overshoot" is the result of a decrease in the influx rate and of an increase in the rate constant of efflux as lactose accumulates. These phenomena were irreversible. The extent of the overshoot was dependent upon the experimental conditions: it was maximal at alkaline pH, for low external potassium concentrations, and for relatively high external lactose concentrations (around or above the KT of uptake). The addition of an energy source to resting E. coli cells resulted in an increase in both the electrochemical gradient of protons and in the rate of respiration. We demonstrate that the overshoot is the result of the latter and unrelated to the former. We observed an irreversible decrease in the membrane potential as lactose accumulated in the presence of an exogenous energy source. We discuss the whole of our data in terms of an irreversible inactivation of the lactose carrier as a result of a possible interaction with the respiratory chain.     

Regulation of squalene epoxidase activity and comparison of catalytic properties of rat liver and Chinese hamster ovary cell-derived enzymes

Squalene epoxidase activity has been studied in cell-free preparations of Chinese hamster ovary (CHO) cells and rat liver. In contrast to rat liver microsomal squalene epoxidase, the enzyme of CHO cells is only slightly activated by the autologous cytosolic fraction, whereas phosphatidylglycerol or rat liver cytosolic preparations are potent stimulators of this enzyme. Triton X-100, a known stimulator of the hepatic squalene epoxidase, has no activating effect on the enzyme of CHO cells. The squalene epoxidase activity of both rat liver and CHO cells varies significantly according to the lipid content of the growth medium or diet. The changes in enzyme activity are shown to be entirely due to altered microsomal enzyme per se and not to changes in the activating properties of the soluble fraction. These results further support the proposed regulatory role of squalene epoxidase in cholesterogenesis.     

Analysis of histones in Xenopus laevis. I. A distinct index of enriched variants and modifications exists in each cell type and is remodeled during developmental transitions

Histone proteins contain epigenetic information that is encoded both in the relative abundance of core histones and variants and particularly in the post-translational modification of these proteins. We determined the presence of such variants and covalent modifications in seven tissue types of the anuran Xenopus laevis, including oocyte, egg, sperm, early embryo equivalent (pronuclei incubated in egg extract), S3 neurula cells, A6 kidney cells, and erythrocytes. We first developed a new robust method for isolating the stored, predeposition histones from oocytes and eggs via chromatography on heparin-Sepharose, whereas we isolated chromatinized histones via conventional acid extraction. We identified two previously unknown H1 isoforms (H1fx and H1B.Sp) present on sperm chromatin. We immunoblotted this global collection of histones with many specific post-translational modification antibodies, including antibodies against methylated histone H3 on Lys(4), Lys(9), Lys(27), Lys(79), Arg(2), Arg(17), and Arg(26); methylated histone H4 on Lys(20); methylated H2A and H4 on Arg(3); acetylated H4 on Lys(5), Lys(8), Lys(12), and Lys(16) and H3 on Lys(9) and Lys(14); and phosphorylated H3 on Ser(10) and H2A/H4 on Ser(1). Furthermore, we subjected a subset of these histones to two-dimensional gel analysis and subsequent immunoblotting and mass spectrometry to determine the global remodeling of histone modifications that occurs as development proceeds. Overall, our observations suggest that each metazoan cell type may have a unique histone modification signature correlated with its differentiation status.     

Insulin-like effects of vanadium: basic and clinical implications

Most mammalian cells contain vanadium at a concentration of about 20 nM, the bulk of which is probably in the reduced vanadyl (+4) form. Although this trace element is essential and should be present in the diet in minute quantities, no known physiological role for vanadium has been found thus far. In the late 1970s the vanadate ion was shown to act as an efficient inhibitor of Na+,K+-ATPase as well as of other related phosphohydrolases. In 1980 vanadium was reported to mimic the metabolic effects of insulin in rat adipocytes. During the last decade, vanadium has been found to act in an insulin-like manner in all three main target tissues of the hormone, namely skeletal muscles, adipose, and liver. Subsequent studies revealed that the action of vanadium salts is mediated through insulin-receptor independent alternative pathway(s). The investigation of the antidiabetic potency of vanadium soon ensued. Vanadium therapy was shown to normalize blood glucose levels in STZ-rats and to cure many hyperglycemia-related deficiencies. Therapeutic effects of vanadium were then demonstrated in type II diabetic rodents, which do not respond to exogenously administered insulin. Finally, clinical studies indicated encouraging beneficial effects. A major obstacle, however, is overcoming vanadium toxicity. Recently, several organically chelated vanadium compounds were found more potent and less toxic than vanadium salts in vivo. Such a newly discovered organic chelator of vanadium is described in this review.