RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.     

Activation of the pro-migratory bone morphogenetic protein receptor 1B gene in human MDA-MB-468 triple-negative breast cancer cells that over-express CYP2J2

Secondary metastases are the leading cause of mortality in patients with breast cancer. Cytochrome P450 (CYP) 2J2 (CYP2J2) is upregulated in many human tumors and generates epoxyeicosanoids from arachidonic acid that promote tumorigenesis and metastasis, but at present there is little information on the genes that mediate these actions. In this study MDA-MB-468 breast cancer cells were stably transfected with CYP2J2 (MDA-2J2 cells) and Affymetrix microarray profiling was undertaken. We identified 182 genes that were differentially expressed in MDA-2J2 cells relative to control (MDA-CTL) cells (log[fold of control] ≥2). From gene ontology pathway analysis bone morphogenetic protein (BMP) receptor 1B (BMPR1B) emerged as an important upregulated gene in MDA-2J2 cells. Addition of the BMPR1B ligand BMP2 stimulated the migration of MDA-2J2 cells, but not MDA-CTL cells, from 3D-matrigel droplets. Migration of MDA-2J2 cells was prevented by the BMPR antagonist dorsomorphin. These findings indicate that over-expression of CYP2J2 in MDA-MB-468-derived breast cancer cells activates BMPR1B expression that may contribute to increased migration. Targeting BMPR1B may be a novel approach to inhibit the metastatic activity of breast cancers that contain high levels of CYP2J2.     

Two fibrinogen-like proteins,FGL1 and FGL2 are disulfide-linked subunits of oligomers that specifically bind nonviable spermatozoa

Nevertheless, a nonviable sperm population is present in the cauda epididymidis of many species. Degenerating spermatozoa release enzymes that could have detrimental effects on the viability of neighboring cells, and they are source of autoantigens that induce an autoimmune response if they escape the blood-epididymis barrier. Does the epididymis have specialized protective mechanism(s) to segregate the viable sperm population from defective spermatozoa? Previously, we identified a fibrinogen-like protein-2 (fgl2) that specifically binds to and polymerizes into a cocoon-like complex coating defective spermatozoa and sperm fragments. The objective of the present study is to identify the subunit composition of the fgl2-containing oligomers both in the soluble and cocoon-like complex. Our proteomic studies indicate that the 260/280 kDa oligomers (termed eFGL) contain two distinct disulfide-linked subunits; 64 kDa fgl2 and 33 kDa fgl1. Utilizing a PCR-based cloning strategy, the 33 kDa polypeptide has been identified as fibrinogen-like protein-1 (fgl1). Immunocytochemical studies revealed that fgl1 selectively binds to defective spermatozoa in the cauda epididymidis. Northern blot analysis and in situ hybridization demonstrated the high expression of fgl1 in the principal cells of the proximal cauda epididymidis. Co-immunoprecipitation analyses of cauda epididymal fluid, using anti-fgl2, demonstrate that both fgl1 and fgl2 are present in the soluble eFGL. Our study is the first to show an association of fgl1 and fgl2 both in the soluble and in the sperm-associated eFGL. We conclude that our results provide new insights into the mechanisms by which the potentially unique epididymal protein functions in the recognition and elimination of defective spermatozoa.     

Vanillin-derived antiproliferative compounds influence Plk1 activity

We synthesized a series of vanillin-derived compounds and analyzed them in HeLa cells for their effects on the proliferation of cancer cells. The molecules are derivatives of the lead compound SBE13, a potent inhibitor of the inactive conformation of human polo-like kinase 1 (Plk1). Some of the new designs were able to inhibit cancer cell proliferation to a similar extent as the lead structure. Two of the compounds ((({4-[(6-chloropyridin-3-yl)methoxy]-3-methoxyphenyl}methyl)(pyridin-4-ylmethyl)amine) and (({4-[(4-chlorophenyl)methoxy]-3-methoxyphenyl}methyl)(pyridin-4-ylmethyl)amine)) were much stronger in their capacity to reduce HeLa cell proliferation and turned out to potently induce apoptosis and reduce Plk1 kinase activity in vitro.     

Evaluation of the antiviral efficacy of bis[1,2]dithiolo[1,4]thiazines and bis[1,2]dithiolopyrrole derivatives against the nucelocapsid protein of the Feline Immunodeficiency Virus (FIV) as a model for HIV infection

A diverse library of bis[1,2]dithiolo[1,4]thiazines and bis[1,2]dithiolopyrrole derivatives were prepared for evaluation of activity against the nucleocapsid protein of the Feline Immunodeficiency Virus (FIV) as a model for HIV, using an in vitro cell culture approach, yielding nanomolar active compounds with low toxicity.     

Proteolytic inactivation of an extracellular (1 → 3)-β-glucanase from the fungus Acremonium persicinum is associated with growth at neutral or alkaline medium pH

Abstract The filamentous fungus Acremonium persicinum released high levels of proteolytic enzyme activity into the culture fluid during growth at pH 7 or above. Almost total inhibition of this crude activity by phenylmethylsulfonyl fluoride suggested that it was mainly due to the presence of a serine protease. This protease inactivated one of three extracellular (1 → 3)- β -glucanases produced by this fungus, although the activities of the remaining two (1 → 3)- β -glucanases did not appear to be affected. Growth of A. persicinum in acidic conditions resulted in the presence of much lower extracellular proteolytic activity and no apparent (1 → 3)- β -glucanase inactivation.     

Endoglucanase A from Cellulomonas fimi in which the hinge sequence of human IgA1 is substituted for the linker connecting its two domains is hydrolyzed by IgA proteases from Neisseria gonorrhoeae

The hinge in IgA1 and the linker in endoglucanase A (CenA) are quite similar. The IgA1 hinge is 18 amino acids long and contains only proline, threonine and serine. The linker in CenA is 27 amino acids long and contains only proline, threonine and a single serine. IgA proteases from Neisseria gonorrhoeae cleave Pro-Ser and Pro-Thr bonds within the IgA1 hinge sequence, but they do not attack CenA. When the linker sequence of CenA is replaced with the hinge sequence of IgA1, the hybrid polypeptide is susceptible to the N. gonorrhoeae proteases. It is cleaved within the hinge sequence at the same sites as IgA1.     

Discovery of isoquinolinone and naphthyridinone-based inhibitors of poly(ADP-ribose) polymerase-1 (PARP1) as anticancer agents: Structure activity relationship and preclinical characterization

The exploitation of GLU988 and LYS903 residues in PARP1 as targets to design isoquinolinone (I & II) and naphthyridinone (III) analogues is described. Compounds of structure I have good biochemical and cellular potency but suffered from inferior PK. Constraining the linear propylene linker of structure I into a cyclopentene ring (II) offered improved PK parameters, while maintaining potency for PARP1. Finally, to avoid potential issues that may arise from the presence of an anilinic moiety, the nitrogen substituent on the isoquinolinone ring was incorporated as part of the bicyclic ring. This afforded a naphthyridinone scaffold, as shown in structure III. Further optimization of naphthyridinone series led to identification of a novel and highly potent PARP1 inhibitor 34, which was further characterized as preclinical candidate molecule. Compound 34 is orally bioavailable and displayed favorable pharmacokinetic (PK) properties. Compound 34 demonstrated remarkable antitumor efficacy both as a single-agent as well as in combination with chemotherapeutic agents in the BRCA1 mutant MDA-MB-436 breast cancer xenograft model. Additionally, compound 34 also potentiated the effect of agents such as temozolomide in breast cancer, pancreatic cancer and Ewing’s sarcoma models.