A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.     

Cutting edge: Autocrine TGF-beta sustains default tolerogenesis by IDO-competent dendritic cells

CD8(-) and CD8(+) dendritic cells (DCs) are distinct subsets of mouse splenic accessory cells with opposite but flexible programs of Ag presentation, leading to immunogenic and tolerogenic responses, respectively. In this study, we show that the default tolerogenic function of CD8(+) DCs relies on autocrine TGF-beta, which sustains the activation of IDO in response to environmental stimuli. CD8(-) DCs do not produce TGF-beta, yet externally added TGF-beta induces IDO and turns those cells from immunogenic into tolerogenic cells. The acquisition of a suppressive phenotype by CD8(-) DCs correlates with activation of the PI3K/Akt and noncanonical NF-kappaB pathways. These data are the first to link TGF-beta signaling with IDO in controlling spontaneous tolerogenesis by DCs.     

Characterization of oversulfated chondroitin sulfate rich in 4,6‐O‐disulfated disaccharides in breast cyst fluids collected from human breast gross cysts

Glycosaminoglycans (GAGs) from breast cyst fluid (BCF) of gross cysts, subdivided into apocrine and flattened, directly collected from 27 gross‐cystic‐breast‐disease (GCBD)‐affected women were analysed. Heparan sulfate, not further investigated, and chondroitin sulfate were identified. This last polysaccharide, in a content of 25–27 µg ml^?1 BCF and having a high molecular mass (~20 000–22 000), was found rich in glucuronic acid (~96%–98%) and mainly sulfated in position 4 of the N‐acetyl‐galactosamine (~60%–64%). Moreover, the presence of ~19%–24% of uncommon 4,6‐O‐disulfated disaccharides CS‐E inside the polysaccharide chains with a high charge density of ~1.15–1.20 was determined. No substantial differences between apocrine and flattened cysts were observed. The current study describes the first effort to examine the yield and distribution of complex macromolecules like GAGs in BCF, and the understanding of their structure may help explain some functions associated with physiological and pathological conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

Taxonomic and biogeographic analysis of the Proasellus coxalis-group (crustacea, isopoda, asellidae) in Sicily, with description of Proasellus montalentii n. sp.

The genus Proasellus is widespread in ponds, ditches and rivers of Sicily. A detailed morphological analysis of several samples of asellids collected in Sicilian freshwaters resulted in the determination of three species of the Proasellus coxalis-group: P. banyulensis italicus (Dudich, 1925), P. montalentii n. sp. and P. wolfi (Dudich, 1925), which is elevated to specific rank. The three species can be distinguished on the basis of the sutures of pleopod V. exopod. Proasellus montalentii inhabits the western part of Sicily, while the range of P. wolfi is confined to the Iblean region. Both species are more closely related to the North African taxa of Proasellus coxalis-group, while Proasellus banyulensis italicus is very similar to the populations found in peninsular Italy. These patterns are explained supposing multiple colonizations of Sicily during Pliocene connections.     

Urinary levels of dehydroepiandrosterone in a group of male patients with functional hyperprolactinemia

On 22 male patients diagnosed as "functional hyperprolactinemia" (the Prolactin (PRL) basal value, was higher than the basal PRL means +/- 2 DS of a control group) we have measured the urinary excretion of Dehydroepiandrosterone (DHEA) mainly produced by adrenal cortex. Our results haven't shown no difference in the urinary excretion of DHEA values in hyperprolactinemic patients has been documented.     

Light-dark cycle and mitotic index in Asellus aquaticus (L.) (Crustacea,Isopoda)

The circadian variation of the mitotic index during spermiohistogenesis was studied in Asellus aquaticus (L.). The actual number of metaphases and prometaphases was determined at the end of each hour of light or darkness over a 24 h period in animals bred under LD 12:12. The number of the metaphases and prometaphases decreases during the light period and sharply increases in the last 3 hrs of the dark period. This variation in the proliferative activity suggests that photoperiod can play a role in the synchronization of mitosis.     

Effects of chemotactic factors and other agents on the amounts of actin and a 65,000-mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils

Stimulation of rabbit neutrophils by the chemotactic factors fMet-Leu-Phe and leukotriene B4, by platelet activating factor, or by arachidonic acid produces a rapid and dose-dependent increase in the amounts of actin and of a 65,000-mol-wt protein associated with the cytoskeleton. Phorbol 12-myristate, 13-acetate, the calcium ionophore A23187 in the presence or absence of EGTA, and the fluorescent calcium chelator quin-2 also cause an increase in cytoskeletal actin. The stimulated increases in the cytoskeletal actin are not dependent on a rise in the intracellular concentration of free calcium and are not mediated by an increase in the intracellular pH or activation of protein kinase C. The increases in the cytoskeletal actin produced by fMet-Leu-Phe and leukotriene B4, but not by phorbol 12-myristate, 13-acetate, are inhibited by high osmolarity. The effect of hyperosmolarity requires a decrease in cell volume, is not mediated by an increase in basal intracellular concentration of free calcium, and is not prevented by pretreating the cells with amiloride. Preincubation of the cells with hyperosmotic solution also inhibits degranulation produced by all the stimuli tested. The inhibitory action of high osmolarity on the fMet-Leu-Phe and leukotriene B4 induced stimulation of cytoskeletal actin is discussed in terms of the possibility that the addition of high osmolarity, either directly or through activation of protein kinase C, causes receptor uncoupling.     

Amino acid ingestion improves muscle protein synthesis in the young and elderly

We recently demonstrated that muscle protein synthesis was stimulated to a similar extent in young and elderly subjects during a 3-h amino acid infusion. We sought to determine if a more practical bolus oral ingestion would also produce a similar response in young (34 +/- 4 yr) and elderly (67 +/- 2 yr) individuals. Arteriovenous blood samples and muscle biopsies were obtained during a primed (2.0 micromol/kg) constant infusion (0.05 micromol.kg(-1).min(-1)) of L-[ring-2H5]phenylalanine. Muscle protein kinetics and mixed muscle fractional synthetic rate (FSR) were calculated before and after the bolus ingestion of 15 g of essential amino acids (EAA) in young (n = 6) and elderly (n = 7) subjects. After EAA ingestion, the rate of increase in femoral artery phenylalanine concentration was slower in elderly subjects but remained elevated for a longer period. EAA ingestion increased FSR in both age groups by approximately 0.04%/h (P < 0.05). However, muscle intracellular (IC) phenylalanine concentration remained significantly higher in elderly subjects at the completion of the study (young: 115.6 +/- 5.4 nmol/ml; elderly: 150.2 +/- 19.4 nmol/ml). Correction for the free phenylalanine retained in the muscle IC pool resulted in similar net phenylalanine uptake values in the young and elderly. EAA ingestion increased plasma insulin levels in young (6.1 +/- 1.2 to 21.3 +/- 3.1 microIU/ml) but not in elderly subjects (3.0 +/- 0.6 to 4.3 +/- 0.4 microIU/ml). Despite differences in the time course of plasma phenylalanine kinetics and a greater residual IC phenylalanine concentration, amino acid supplementation acutely stimulated muscle protein synthesis in both young and elderly individuals.     

Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All

A new discontinuous agarose-gel electrophoresis in 0.05 M HCl/0.04 M barium acetate combined with the highly sensitive visualization technique using toluidine blue/Stains-All has been developed for the simultaneous assaying of hyaluronic acid (HA) and dermatan sulfate (DS) with a detection limit at submicrogram level greater than other conventional procedures. Furthermore, this procedure also separates and reveals chondroitin sulfate (CS). The densitometric analysis of bands resulted in a linear response between 0.01 and 0.5 microg of glycosaminoglycans (GAGs) with correlation coefficients greater than approximately 0.94. Hyaluronic acid and dermatan sulfate extracted and purified from the abdominal skin of six rats were separated and quantified in comparison with the evaluation made by treatment of chondroitin ABC lyase and separation of Delta-disaccharides from hyaluronic acid (DeltadiHA) and dermatan sulfate/chondroitin sulfate (Deltadi4s and Deltadi6s) by HPLC. The total amount of rat skin polysaccharides (hyaluronic acid and dermatan sulfate) was 1.24+/-0.26 microg/mg of tissue by discontinuous agarose-gel electrophoresis and 1.20+/-0.33 microg/mg by HPLC with hyaluronic acid and dermatan sulfate percentages of 50.32+/-2.38 and 49.66+/-2.53, respectively. The analyses also confirmed that hyaluronic acid and dermatan sulfate are the main rat abdominal skin polysaccharides with chondroitin sulfate present in trace amounts. This new agarose-gel electrophoresis could be particularly useful in the study of the distribution of glycosaminoglycans in the skin from different body sites of animals and normal human subjects and may be of importance in understanding the changes that occur in the skin, especially the metabolism of extracellular matrix constituents, in connective tissue disorders.