Pyridyl aminothiazoles as potent inhibitors of Chk1 with slow dissociation rates

Pyridyl aminothiazoles comprise a novel class of ATP-competitive Chk1 inhibitors with excellent inhibitory potential. Modification of the core with ethylenediamine amides provides compounds with low picomolar potency and very high residence times. Investigation of binding parameters of such compounds using X-ray crystallography and molecular dynamics simulations revealed multiple hydrogen bonds to the enzyme backbone as well as stabilization of the conserved water molecules network in the hydrophobic binding region.     

Exercise Improves Body Fat,Lean Mass,and Bone Mass in Breast Cancer Survivors

Given the negative effects of a breast cancer diagnosis and its treatments on body weight and bone mass, we investigated the effects of a 6‐month randomized controlled aerobic exercise intervention vs. usual care on body composition in breast cancer survivors. Secondary aims were to examine the effects stratified by important prognostic and physiologic variables. Seventy‐five physically inactive postmenopausal breast cancer survivors were recruited through the Yale–New Haven Hospital Tumor Registry and randomly assigned to an exercise (n = 37) or usual care (n = 38) group. The exercise group participated in 150 min/week of supervised gym‐ and home‐based moderate‐intensity aerobic exercise. The usual care group was instructed to maintain their current physical activity level. Body composition was assessed at baseline and 6‐months through dual‐energy X‐ray absorptiometry (DXA) by one radiologist blinded to the intervention group of the participants. On an average, exercisers increased moderate‐intensity aerobic exercise by 129 min/week over and above baseline levels compared with 45 min/week among usual care participants (P < 0.001). Exercisers experienced decreases in percent body fat (P = 0.0022) and increases in lean mass (P = 0.047) compared with increases in body fat and decreases in lean mass in usual care participants. Bone mineral density (BMD) was also maintained among exercisers compared with a loss among usual care participants (P = 0.043). In summary, moderate‐intensity aerobic exercise, such as brisk walking, produces favorable changes in body composition that may improve breast cancer prognosis.     

Desensitization of the Neurokinin-1 Receptor (NK1-R) in Neurons: Effects of Substance P on the Distribution of NK1-R, Gαq/11, G-Protein Receptor Kinase-2/3, and β-Arrestin-1/2

Observations in reconstituted systems and transfected cells indicate that G-protein receptor kinases (GRKs) and β-arrestins mediate desensitization and endocytosis of G-protein–coupled receptors. Little is known about receptor regulation in neurons. Therefore, we examined the effects of the neurotransmitter substance P (SP) on desensitization of the neurokinin-1 receptor (NK1-R) and on the subcellular distribution of NK1-R, G_αq/11, GRK-2 and -3, and β-arrestin-1 and -2 in cultured myenteric neurons. NK1-R was coexpressed with immunoreactive G_αq/11, GRK-2 and -3, and β-arrestin-1 and -2 in a subpopulation of neurons. SP caused 1) rapid NK1-R–mediated increase in [Ca²⁺]_i, which was transient and desensitized to repeated stimulation; 2) internalization of the NK1-R into early endosomes containing SP; and 3) rapid and transient redistribution of β-arrestin-1 and -2 from the cytosol to the plasma membrane, followed by a striking redistribution of β-arrestin-1 and -2 to endosomes containing the NK1-R and SP. In SP-treated neurons G_αq/11 remained at the plasma membrane, and GRK-2 and -3 remained in centrally located and superficial vesicles. Thus, SP induces desensitization and endocytosis of the NK1-R in neurons that may be mediated by GRK-2 and -3 and β-arrestin-1 and -2. This regulation will determine whether NK1-R–expressing neurons participate in functionally important reflexes.     

Root biomass allocation in the world's upland forests

Because the world's forests play a major role in regulating nutrient and carbon cycles, there is much interest in estimating their biomass. Estimates of aboveground biomass based on well-established methods are relatively abundant; estimates of root biomass based on standard methods are much less common. The goal of this work was to determine if a reliable method to estimate root biomass density for forests could be developed based on existing data from the literature. The forestry literature containing root biomass measurements was reviewed and summarized and relationships between both root biomass density (Mg ha⁻¹) and root:shoot ratios (R/S) as dependent variables and various edaphic and climatic independent variables, singly and in combination, were statistically tested. None of the tested independent variables of aboveground biomass density, latitude, temperature, precipitation, temperature:precipitation ratios, tree type, soil texture, and age had important explanatory value for R/S. However, linear regression analysis showed that aboveground biomass density, age, and latitudinal category were the most important predictors of root biomass density, and together explained 84% of the variation. A comparison of root biomass density estimates based on our equations with those based on use of generalized R/S ratios for forests in the United States indicated that our method tended to produce estimates that were about 20% higher. Received: 3 July 1996 / Accepted: 23 January 1997     

Gangliosides and β1‐Integrin Are Required for Caveolae and Membrane Domains

Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin‐mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo‐glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol (‘lipid rafts'), reduced caveolae and caveolin‐1 at the plasma membrane by approximately 80%, and blunted activation of β1‐integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo‐gangliosides or other sphingolipids) at 10°C, suggesting that sialo‐lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Iγ away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin‐1 could be recapitulated by β1‐integrin knockdown. These results suggest that both gangliosides and β1‐integrin are required for maintenance of caveolae and plasma membrane domains.     

ePAD,an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets

Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.