High-level expression of active HIV-1 integrase from a synthetic gene in human cells.

A synthetic gene encoding for HIV-1 integrase was designed to circumvent the intrinsic instability and the repressor elements present in the wild-type gene. High-level expression of HIV-1 integrase was obtained in various human cell lines independently of viral accessory proteins. A human 293T cell line was selected that stably expresses HIV-1 integrase and has growth kinetics comparable to the parental cell line. The enzyme was localized in the nucleus and remained stably associated with the chromosomes during mitosis. Lentiviral vector particles carrying the inactivating D64V mutation in the integrase gene were capable of stably transducing 293T cells when complemented in the producer cells with integrase expressed from the synthetic gene. When the cell line that stably expresses integrase was infected with the defective viral particles, complementation of integrase activity was detected as well. Expression of active HIV-1 integrase in human cells will facilitate the study of the interplay between host and viral factors during integration.     

Binding of nucleotides by T4 DNA ligase and T4 RNA ligase: optical absorbance and fluorescence studies.

The interaction of nucleotides with T4 DNA and RNA ligases has been characterized using ultraviolet visible (UV-VIS) absorbance and fluorescence spectroscopy. Both enzymes bind nucleotides with the K(d) between 0.1 and 20 microM. Nucleotide binding results in a decrease of absorbance at 260 nm due to pi-stacking with an aromatic residue, possibly phenylalanine, and causes red-shifting of the absorbance maximum due to hydrogen bonding with the exocyclic amino group. T4 DNA ligase is shown to have, besides the catalytic ATP binding site, another noncovalent nucleotide binding site. ATP bound there alters the pi-stacking of the nucleotide in the catalytic site, increasing its optical extinction. The K(d) for the noncovalent site is approximately 1000-fold higher than for the catalytic site. Nucleotides quench the protein fluorescence showing that a tryptophan residue is located in the active site of the ligase. The decrease of absorbance around 298 nm suggests that the hydrogen bonding interactions of this tryptophan residue are weakened in the ligase-nucleotide complex. The excitation/emission properties of T4 RNA ligase indicate that its ATP binding pocket is in contact with solvent, which is excluded upon binding of the nucleotide. Overall, the spectroscopic analysis reveals important similarities between T4 ligases and related nucleotidyltransferases, despite the low sequence similarity.     

Nature of the turtle shell: Morphogenetic causes of bone variability and its evolutionary implication

The turtle shell is characterized by a high degree of conservatism of the fundamental model and, at the same time, a high variability at the individual level. The components of the bony shell vary in origin. The costal and neural plates of the carapace are modified elements of the axial skeleton (ribs and neural arches) and the plastral plates are transformed dermal ossifications of the shoulder girdle and gastralia, peripheral, pygal, and suprapygal plates are similar to osteoderms of other reptiles. The variability of the structure of particular parts of the turtle shell is manifested differently. Most anomalies have been recorded in the caudal part of the carapace. The plastron is relatively stable in morphology. Variations in the bony shell structure are observed in (1) unusual shape and size of plates combined with normal number of plates, (2) presence of additional plates, and (3) absence of regular plates. Based on the morphogenetic characteris-tics, anomalies are subdivided into (1) variations caused by changes in the number of elements of the axial skeleton or their contacts with the dermis (neurals and costals); (2) variations due to changes in the number of horny scutes (peripherals); (3) variations connected with irregular osteogeny or disturbed growth of bones     

Computational and experimental studies of the electronic excitation spectra of EDTA and DTPA substituted tetraphenylporphyrins and their Lu complexes

Ethylendiaminetetraacetic acid (EDTA) substituted and diethylenetriaminopentaacetic acid (DTPA) substituted aminated free-base tetraphenylporphyrins (H₂ATPP) and the corresponding lutetium(III) complexes have been studied computationally at the density functional theory (DFT) and second-order algebraic diagrammatic construction (ADC(2)) levels using triple-ξ basis sets augmented with polarization functions. The molecular structures were optimized using Becke's three-parameter hybrid functional (B3LYP). The electronic excitation spectra in the range of 400–700 nm were calculated using the ADC(2) and the linear-response time-dependent DFT methods. The calculated spectra are compared to those measured in ethanol solution. The calculated excitation energies agree well with those deduced from the experimental spectra. The excitation energies for the Q_x band calculated at the B3LYP and ADC(2) level are 0.20-0.25 eV larger than the experimental values. The excitation energies for the Q_y band calculated at the B3LYP level are 0.10-0.20 eV smaller than the ADC(2) ones and are thus in good agreement with experiment. The calculated excitation energies corresponding to the B_x and B_y bands are 0.10-0.30 eV larger than the experimental values. The excitation energies of the B_x and B_y bands calculated at the B3LYP level are in somewhat better agreement with experiment than the ADC(2) ones. The calculated and measured band strengths largely agree.

Structural insights into the retroviral DNA integration apparatus

Retroviral replication depends on successful integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 3' viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes.     

Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75

Human lens epithelium-derived growth factor (LEDGF)/p75 protein forms a specific nuclear complex with human immunodeficiency virus type 1 (HIV-1) integrase and is essential for nuclear localization and chromosomal association of the viral protein. We now studied nuclear import of LEDGF/p75 in live and semipermeabilized cells. We show that nuclear import of LEDGF/p75 is GTP-, Ran-, importin-alpha/beta-, and energy-dependent and that the protein competes with the canonical SV40 large T antigen nuclear localization signal (NLS) for nuclear import receptors. We identified the NLS of LEDGF/p75 through deletion analysis and site-directed mutagenesis. The LEDGF/p75 NLS, 148GRKRKAEKQ156, belongs to the canonical SV40-like family. Fusion of this short peptide to the amino terminus of Escherichia coli beta-galactosidase rendered the fusion protein nuclear, confirming that the LEDGF/p75 NLS is transferable. Moreover, a single amino acid change in the NLS was sufficient to exclude the mutant LEDGF/p75 protein from the nucleus and abolish nuclear import of HIV-1 integrase.     

Insulin, insulin-like growth factor-I, and platelet-derived growth factor activate extracellular signal-regulated kinase by distinct pathways in muscle cells

We have investigated the signaling pathways initiated by insulin, insulin-like growth factor-1 (IGF-I), and platelet-derived growth factor (PDGF) leading to activation of the extracellular signal-regulated kinase (ERK) in L6 myotubes. Insulin but not IGF-I or PDGF-induced ERK activation was abrogated by Ras inhibition, either by treatment with the farnesyl transferase inhibitor FTP III, or by actin disassembly by cytochalasin D, previously shown to inhibit Ras activation. The protein kinase C (PKC) inhibitor bisindolylmaleimide abolished PDGF but not IGF-I or insulin-induced ERK activation. ERK activation by insulin, IGF-I, or PDGF was unaffected by the phosphatidylinositol 3-kinase inhibitor wortmannin but was abolished by the MEK inhibitor PD98059. In contrast, activation of the pathway involving phosphatidylinositol 3-kinase (PI3k), protein kinase B, and glycogen synthase kinase 3 (GSK3) was mediated similarly by all three receptors, through a PI 3-kinase-dependent but Ras- and actin-independent pathway. We conclude that ERK activation is mediated by distinct pathways including: (i) a cytoskeleton- and Ras-dependent, PKC-independent, pathway utilized by insulin, (ii) a PKC-dependent, cytoskeleton- and Ras-independent pathway used by PDGF, and (iii) a cytoskeleton-, Ras-, and PKC-independent pathway utilized by IGF-I.     

Transient accumulation of elastic energy in proton translocating ATP synthase

ATP synthase is conceived as a rotatory engine with two reversible drives, the proton-transporting membrane portion, F0, and the catalytic peripheral portion, F1. They are mounted on a central shaft (subunit gamma) and held together by an eccentric bearing. It is established that the hydrolysis of three molecules of ATP in F1 drives the shaft over a full circle in three steps of 120 degrees each. Proton flow through F0 probably generates a 12-stepped rotation of the shaft so that four proton-translocating steps of 30 degrees each drive the synthesis of one molecule of ATP. We addressed the elasticity of the transmission between F0 and F1 in a model where the four smaller steps in F0 load a torsional spring which is only released under liberation of ATP from F1. The kinetic model of an elastic ATP synthase described a wealth of published data on the synthesis/hydrolysis of ATP by F0F1 and on proton conduction by F0 as function of the pH and the protonmotive force. The pK values of the proton-carrying group interacting with the acidic and basic sides of the membrane were estimated as 5.3-6.4 and 8.0-8.3, respectively.     

Electron transfer through the acceptor side of photosystem I: Interaction with exogenous acceptors and molecular oxygen

This review considers the state-of-the-art on mechanisms and alternative pathways of electron transfer in photosynthetic electron transport chains of chloroplasts and cyanobacteria. The mechanisms of electron transport control between photosystems (PS) I and II and the Calvin–Benson cycle are considered. The redistribution of electron fluxes between the noncyclic, cyclic, and pseudocyclic pathways plays an important role in the regulation of photosynthesis. Mathematical modeling of light-induced electron transport processes is considered. Particular attention is given to the electron transfer reactions on the acceptor side of PS I and to interactions of PS I with exogenous acceptors, including molecular oxygen. A kinetic model of PS I and its interaction with exogenous electron acceptors has been developed. This model is based on experimental kinetics of charge recombination in isolated PS I. Kinetic and thermodynamic parameters of the electron transfer reactions in PS I are scrutinized. The free energies of electron transfer between quinone acceptors A_1A/A_1B in the symmetric redox cofactor branches of PS I and iron–sulfur clusters F_X, F_A, and F_B have been estimated. The second-order rate constants of electron transfer from PS I to external acceptors have been determined. The data suggest that byproduct formation of superoxide radical in PS I due to the reduction of molecular oxygen in the A1 site (Mehler reaction) can exceed 0.3% of the total electron flux in PS I.