Depletion of plasma membrane-associated phosphoinositides mimics inhibition of TRPM7 channels by cytosolic Mg, spermine, and pH.

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is an ion channel/protein kinase belonging to the TRP melastatin and eEF2 kinase families. Under physiological conditions, most native TRPM7 channels are inhibited by cytoplasmic Mg, protons, and polyamines. Currents through these channels (I) are robustly potentiated when the cell interior is exchanged with low Mg-containing buffers.

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway.

Hydroxyurea (HU) has been used for the treatment of multiple diseases, such as cancer. The therapeutic effect is generally believed to be due to the suppression of ribonucleotide reductase (RNR), which slows DNA polymerase movement at replication forks and induces an S phase cell cycle arrest in proliferating cells. Although aberrant mitosis and DNA damage generated at collapsed forks are the likely causes of cell death in the mutants with defects in replication stress response, the mechanism underlying the cytotoxicity of HU in wild-type cells remains poorly understood.

A single amino acid change humanizes long-chain fatty acid binding and activation of mouse peroxisome proliferator-activated receptor α.

Peroxisome proliferator-activated receptor α (PPARα) is an important regulator of hepatic lipid metabolism which functions through ligand binding. Despite high amino acid sequence identity (>90%), marked differences in PPARα ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we have recently reported differences in ligand affinities and extent of activation between human PPARα (hPPARα) and mouse PPARα (mPPARα) in response to long chain fatty acids (LCFA).

Canonical Bcl-2 motifs of the Na+/K+ pump revealed by the BH3 mimetic chelerythrine: early signal transducers of apoptosis?

Background/aims: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs].

Methods: K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC.

Prediction of and experimental support for the three-dimensional structure of replication protein A.

Replication protein A (RPA) is a heterotrimeric, multidomain, single-stranded DNA binding protein that is essential for DNA replication, repair, and recombination. Crystallographic and NMR studies on RPA protein fragments have provided structures for all domains; however, intact heterotrimeric RPA has resisted crystallization, and a complete protein structure has not yet been described. In this study, computational methods and experimental reactivity information (MRAN) were used to model the complete structure of RPA.

Reactivity-based analysis of domain structures in native replication protein A.

Replication protein A (RPA) is an essential heterotrimeric ssDNA binding protein that participates in DNA repair, replication, and recombination. Though X-ray and NMR experiments have been used to determine three-dimensional structure models of the protein's domain fragments, a complete RPA structural model has not been reported. To test whether the fragment structures faithfully represent the same portions in the native solution-state protein, we have examined the structure of RPA under biologically relevant conditions.

DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage.

Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies.

Denaturation of replication protein A reveals an alternative conformation with intact domain structure and oligonucleotide binding activity.

Replication protein A (RPA) is a heterotrimeric, multidomain, single-stranded DNA-binding protein. Using spectroscopic methods and methylene carbene-based chemical modification methods, we have identified conformational intermediates in the denaturation pathway of RPA. Intrinsic protein fluorescence studies reveal unfolding profiles composed of multiple transitions, with midpoints at 1.

The reverse transcriptase sequence of human immunodeficiency virus type 1 is under positive evolutionary selection within the central nervous system.

The human immunodeficiency virus type 1 (HIV-1) enters the central nervous system (CNS) during the acute phase of infection and causes AIDS-related encephalitis and dementia in 30% of individuals. Previous studies show that HIV-1 sequences derived from the CNS of infected patients, including the sequence encoding reverse transcriptase (RT), are genetically distinct from sequences in other tissues. The hypothesis of the current study is that the RT sequence of HIV-1 is under positive selection within the CNS.