Kinetics of the dimerization of retroviral proteases: the "fireman's grip" and dimerization

All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the "fireman's grip," which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp-Thr(Ser)-Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp-Thr-Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild-type and mutant proteases from HIV and myeloblastosis-associated virus harboring either Ser or Thr in their Asp-Thr(Ser)-Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be approximately 10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.     

Telomere variability in the monocotyledonous plant order Asparagales

A group of monocotyledonous plants within the order Asparagales, forming a distinct clade in phylogenetic analyses, was reported previously to lack the 'typical' Arabidopsis-type telomere (TTTAGGG)(n). This stimulated us to determine what has replaced these sequences. Using slot-blot and fluorescent in situ hybridization (FISH) to species within this clade, our results indicate the following. 1. The typical Arabidopsis-type telomeric sequence has been partly or fully replaced by the human-type telomeric sequence (TTAGGG)(n). Species in Allium lack the human-type variant. 2. In most cases the human variant occurs along with a lower abundance of two or more variants of the minisatellite sequences (of seven types evaluated), usually these being the consensus telomeric sequence of Arabidopsis, Bombyx (TTAGG)(n) and Tetrahymena (TTGGGG)(n). FISH shows that the variants can occur mixed together at the telomere. 3. Telomerases generate products with a 6 base pair periodicity and when sequenced they reveal predominantly a reiterated human-type motif. These motifs probably form the 'true telomere' but the error rate of motif synthesis is higher compared with 'typical' plant telomerases. The data indicate that the Asparagales clade is unified by a mutation resulting in a switch from synthesis of Arabidopsis-like telomeres to a low-fidelity synthesis of human-like telomeres.     

(S)-2-chloro-2-fluoroethanoyl isocyanate,a chiral derivative of trichloroacetyl isocyanate

Carbamate diastereomers 3b-18b were prepared from easily accessible (S)-2-chloro-2-fluoroethanoyl isocyanate (1) and various secondary chiral alcohols. Compound 1 as a chiral analog of trichloroacetyl isocyanate undergoes the reaction with alcohols very fast, thus blocking the hydroxyl group for the purposes of NMR investigation. Moreover, the correlation of stereochemistry of 3b-18b with their (1)H NMR spectra revealed that the constitution as well as configuration influences regularly the values of chemical shift difference (deltadelta = delta(R) - delta(S)) except for those diastereomers bearing simple alkyl groups in the molecule. Spectral as well as crystallographic data manifest the predominant planar conformation of the central part of the molecule. Due to the good accessibility and high reactivity in particular, the acylisocyanate 1 might be considered, to some extent, an alternative for TAI giving additional information on a compound's spatial structure.     

Pathogenesis of prosthesis-related infection

In spite of its incidence decreasing to 1% nowadays, prosthesis-related infection remains a research, diagnostic, therapeutic and cost-related problem. It can be defined as a presence of bacteria in the artificial joint space, which is significantly associated with evident laboratory and/or tissue markers, and clinical signs of running infection. We believe that the more precise understanding of pathogenesis, the more effective preventative and therapeutic measures, and the lower infection rate. The implants are colonized by airborne, skin-, and/ or surgeon-related bacteria during surgery despite being operated in closely respected operating regime. Some prosthetic characteristics are advantageous and may play important roles in the process of bacterial adherence. After successful attachment on the biomaterial surface bacteria multiply and physiologically transform into a "biofilm" community, making them much more resistant to antibiotic therapy and host immunity. Bacterial resistance is a complex phenomenon influenced by intrinsic and extrinsic factors, including the cell configuration in the biofilm community. So the cure of periprosthetic sepsis without removing of all foreign bodies and necrotic bone fragments is often ineffective. Acute hematogenous sepsis is suggestive of a distortion of a previously aseptic joint space by invasion of bacteria through the vessels.     

Study on reactivity and protection of the alpha-hydroxyphosphonate moiety in 5'-nucleotide analogues: formation of the 3'-O-P-C(OH)-C4' internucleotide linkage

The recently described epimeric nucleosidyl-5'-C-phosphonates (alpha-hydroxyphosphonates) represent novel nucleotide analogues that can be incorporated into chimeric oligonucleotides by the phosphotriester condensation method. In order to prepare suitable protected monomer(s) we have studied condensation reaction between protected 2'-deoxythymidine and 2'-deoxythymidinyl-5'-C-phosphonate, both as model compounds, in dependence on the nature of the 5'-hydroxyl protecting group. We have found that the O-acetyl group is unstable in the presence of TPSCl or MSNT used as condensing agents for activation of the phosphorus moiety. This instability negatively influences the scope of the condensation process. On the other hand, introduction of the O-methoxycarbonyl group gave excellent results. The O-methoxycarbonyl group does not participate in the condensation process, and its quantitative introduction into the nucleotide analo gues is accomplished using a novel acylating agent, methoxycarbonyl tetrazole.     

Ribo-, xylo-, and arabino-configured adenine-based nucleoside phosphonates: synthesis of monomers for solid-phase oligonucleotide assembly

Adenine-based, regioisomeric nucleoside phosphonates with ribo, xylo and arabino configuration were synthesized in the protected form suitable for the phosphotriester-like, solid-phase synthesis of oligonucleotides. Phosphonate moiety was protected by 4-methoxy-1-oxido-2-picolyl group and the furanose hydroxyl by the dimethoxytrityl group.     

Swelling pressure induced phase-volume transition in hybrid biopolymer gels caused by unfolding of folded crosslinks: a model

A thermodynamic model is proposed describing swelling changes and swelling transitions of hybrid gels in which domains of folded chains are chemically built in as cross-links. These folded domains can be unfolded to random coils by osmotic forces produced by the synthetic gel matrix. Uncoiling takes place if the osmotic force acting on the cross-links exceeds the critical value. By unfolding, a new interacting surface is exposed to interactions and affects the swelling pressure. The chains of the folded domains may have ionized groups. The model is based on mean-field statistical-thermodynamic treatment of swelling of polyelectrolyte gels with finite extensibility of network chains. This study is related to hybrid hydrogels with built in protein motifs. A continuous change in external variables increasing the degree of swelling of the hydrogel brings about an abrupt increase in volume (transition) of the gel. The position and magnitude of the transition depend on structural parameters of the hybrid gel, such as fraction of the folded domains in the gel, degree of ionization of chains in the domain, presence of additional chemical cross-links, or degree of dilution at gel formation. Two options for reversibility of the changes are considered: (a) unfolding is irreversible and deswelling proceeds along other curve than swelling and (b) swelling is reversible when the osmotic force decrease below the critical value. In the latter case, swelling changes are described by a closed loop with two transitions. Under certain conditions (high dilution at network formation and sufficiently high degree of ionization of chains of the folded domains), a transition appears known as the collapse transition induced by balance of hydrophobic and hydrophillic interactions. This collapse transition induces the folding transition by which the folded domains are reformed.     

Inverse relationship between serum bilirubin and atherosclerosis in men: a meta-analysis of published studies

Bilirubin, a major intravascular product of heme catabolism, is a potent antioxidant compound. Numerous studies have been published showing the relationship between serum bilirubin levels and atherosclerosis. In the present investigation all the epidemiological studies available on the effect of serum bilirubin levels and atherosclerotic disease were analyzed. Studies on the epidemiology of atherosclerotic diseases in relation to serum bilirubin levels were searched in the MEDLINE database. Selected studies were subdivided according to serum bilirubin levels and severity of atherosclerotic disease. Because of the limited number of females involved in the studies, only males were included into meta-analysis. Associations for ordered categorical variables (bilirubin and natural history of graded atherosclerosis) were assessed to find correlation and linear trend between analyzed variables. A stratified analysis was conducted to compare risks of clinical outcomes. Eleven relevant studies were used for analysis. A close negative relationship was found between serum bilirubin levels and severity of atherosclerosis (Spearman rank coefficient r = -0.31,P < 0.0001). The linear trend was confirmed in analysis of proportions with x(2) values for both disease conditions to be very significant (P < 0.0001). Unambiguous inverse relationship between serum bilirubin levels and atherosclerosis was demonstrated in this preliminary meta-analytic study. These results indicate the importance of hem oxygenase-related products in the prevention of oxidative stress-mediated diseases.     

Modification of activity and specificity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 by engineering of its entrance tunnel

Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.