Multiple modes of ligand recognition: Crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors,olomoucine and isopentenyladenine

Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(δ2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties. © 1995 Wiley-Liss, Inc.     

pAMβ1 resolvase has an atypical recombination site and requires a histone-like protein HU

The broad-host-range plasmid pAMβ1 from Gram-positive bacteria encodes a resolvase, designated Resβ, which shares homology with the proteins of the resolvase—invertase family. Here we report the purification and in vitro characterization of Resβ. This resolvase is particular in two aspects: it has an atypical binding site and requires a cofactor to promote resolution in vitro . Resβ binds to two regions within its resolution site res . One contains two inverted repeats (R1 and R2), the other contains only one repeat (R3). The cofactor required for resolution in vitro is present in crude extracts of both Bacillus subtilis and Escherichia coli and can be substituted by the E. coli histone-like protein HU. The possible mode of action of HU in the resolution process is discussed.     

A three-step purification of human alpha 1-acid glycoprotein

alpha 1-Acid glycoprotein (AGP) was purified to homogeneity by a 3-step procedure using pseudo-ligand affinity chromatography on immobilized Cibacron blue F3GA, Procion red HE3B, and preparative column isoelectric focusing. The overall yield of the combined techniques was 88%. Analysis of the purified AGP by lectin affinity chromatography on immobilized Con A and immunoaffino-electrophoresis indicated that the most acidic form did not interact with the lectin, while the two more basic fractions possessed different affinities for Con A. In addition, 3 different populations of AGP were clearly separated by Con A affinity chromatography.     

Studies in vitro on the uptake and degradation of sodium hyaluronate in rat liver endothelial cells.

Rat liver endothelial cells in primary cultures at 7 degrees C bind radioactively labelled sodium hyaluronate (HA; Mr 400 000) specifically and with high affinity (Kd = 6 X 10(-11) M). Maximal binding capacity is approx. 10(4) molecules per cell. Inhibition experiments with unlabelled HA and oligosaccharides from HA indicate that each molecule is bound by several receptors acting co-operatively and that the single receptor recognizes a tetra- or hexa-saccharide sequence of the polysaccharide. At 37 degrees C the liver endothelial cells endocytose the HA. The process combines the features of a receptor-mediated and a fluid-phase endocytosis. The rate of internalization does not show any saturation with increasing HA concentration, but is approximately proportional to the polysaccharide concentration at and above the physiological concentration. At 50 micrograms of free HA/l each liver endothelial cell accumulates 0.1 fg of the polysaccharide/min. Fluorescent HA accumulates in perinuclear granules, presumably lysosomes. Degradation products from HA appear in the medium about 30 min after addition of the polysaccharide to the cultures. The radioactivity from HA containing N-[3H]acetyl groups or 14C in the sugar rings is recovered mainly as [3H]acetate and [14C]acetate respectively. Estimations of the capacity of liver endothelial cells to internalize and degrade HA in vitro indicate that these cells may be primarily responsible for the clearance of HA from human blood in vivo.     

Stereometry of the red pulp on the human spleen. I) Methodological remarks

The principal histometrical formulas to be applied to the analysis of human spleen's red pulp are illustrated. The discussion particularly concerns the formulas to evaluate the following measures per unit splenic volume:volume of the elements; surface area of sinus-splenic cord boundaries; mean breadth of cords; length of the sinus; mean sectional area of the sinus. Suggestions are given about the criteria to be followed in the proper assumption of the data which must be subjected to statistical estimation.     

A new enkephalin analogue: trans-4-hydroxycinnamoyl-glycyl-glycyl-phenylalanyl-leucine. Synthesis and biological properties

A new analogue of the leucine-enkephalin in which the N-terminal tyrosine has been replaced by trans-4-hydroxycinnamic acid, has been synthetized by liquid-phase coupling methods. The central cardiovascular effects of this analogue were investigated and the results discussed.     

Concurrent or sequential use of cytotoxic chemotherapy and hormone treatment in advanced breast cancer: report of the Swiss Group for Clinical Cancer Research.

In a trial of combined hormone treatment and cytotoxic chemotherapy 464 patients with advanced breast cancer were randomly allocated to either concurrent or sequential treatment. Cytotoxic drugs were given only if the antitumour activity of the hormone treatment was inadequate. Hormone treatment consisted of oophorectomy for premenopausal and tamoxifen administration for postmenopausal patients. Length of survival was better, though not significantly, in premenopausal patients (p = 0.29) treated concurrently and in postmenopausal women (p = 0.17) treated sequentially; the difference was highly significant (p = 0.003) only for postmenopausal women in the low-risk category. The findings suggest that postmenopausal women with metastatic breast cancer should probably be treated primarily by carefully monitored hormone treatment.     

Uptake of hyaluronan in hepatic endothelial cells is not directly affected by endotoxin and associated cytokines

The uptake of hyaluronan (HYA) labeled with 3H in its acetyl group was measured in cultured liver endothelial cells from normal rats and from rats previously treated with sublethal doses of Escherichia coli endotoxin (ET). Replicate cultures were also exposed to recombinant human tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or interferon-gamma for 1 to 3 h before the measurement of hyaluronan uptake. Under all conditions, HYA was absorbed by endothelial cells at rates consistent with receptor-mediated absorption. In cells exposed to HYA 20 h after isolation, rate of uptake was less than half the rate in cells exposed 6 or 7 h after isolation. Cellular uptake of HYA was neither reduced nor enhanced by any of the treatments with cytokines. Prior exposure of the cell donors to ET caused a three-fold increase in their plasma HYA but did not alter the subsequent rate of cellular HYA uptake in vitro, either with or without added treatment with TNF-alpha or IL-1. It was concluded that the elevation of plasma HYA caused by septicaemia or by the experimental administration of ET or TNF-alpha cannot be attributed to direct interference with HYA receptors on hepatic endothelial cells.     

Ligand-dependent secretion of rat retinol-binding protein expressed in HeLa cells.

A minigene encoding rat retinol-binding protein (RBP) was transfected into HeLa cells, which do not express endogenous RBP, transthyretin, or cellular retinol-binding protein. The HeLa cells manufactured and secreted the transfected gene product, demonstrating that RBP-transthyretin assembly is not a requirement for the secretion of RBP. When HeLa cells were grown under vitamin A-deficient conditions, RBP accumulated in the endoplasmic reticulum. Both serum and retinol stimulated secretion of RBP in a concentration-dependent manner. The retinol-regulated secretion occurred also after protein synthesis had been blocked by cycloheximide. Addition of holo-RBP or retinal, but not retinoic acid, stimulated secretion of RBP. Thus, an in vitro model system that resembles the rat hepatocyte in vivo with regard to the known regulation of RBP secretion has been established in a human cell line of extrahepatic origin. It can be concluded that cellular retinol-binding protein is not required for the transfer of retinol to RBP and that the mechanism whereby retinol controls the intracellular transport of RBP is neither specific for tissues synthesizing RBP nor species-specific. To investigate the structural properties responsible for the endoplasmic reticulum retention of RBP in the absence of its ligand, a cDNA encoding chicken purpurin, a protein that is 50% identical to RBP and that binds retinol, was expressed in HeLa cells. In contrast to RBP, purpurin was not retained in vitamin A-deficient HeLa cells.