Activity of paraoxonase 1 (PON1) and its relationship to markers of lipoprotein oxidation in healthy Slovaks

Low-density lipoproteins (LDLs), when modified by free radicals derived from artery wall cells, induce atherosclerosis. In contrast to oxidized LDL (ox-LDL), high-density lipoproteins (HDLs) are able to prevent atherosclerosis through a protein with antioxidant properties, paraoxonase 1 (PON1). The purpose of this study was to explore the association between the activity of HDL-associated PON1 and circulating ox-LDL as well as to investigate the relationship between ox-LDL and parameters of lipid profile in thirty Slovaks aged 21-73 years because recent studies have presented controversial results concerning PON1 and its role in LDL oxidation. For determination of circulating ox-LDL sandwich ELISA was used and other lipid parameters were determined by routine laboratory analyses. PON1 activities were assayed by two synthetic substrates - paraoxon and phenyl acetate. Lipid peroxides were determined spectrophotometrically. Of the lipid parameters examined, ox-LDL level correlated positively with total (P < 0.0001) and LDL-cholesterol (P < 0.001). Triacylglycerols (TAG) (P < 0.001), lipid peroxides (P < 0.01) and atherogenic index (AI = total cholesterol/HDL) (P < 0.0001) were also strongly correlated with ox-LDL. No inverse relationships were observed between ox-LDL and HDL-cholesterol or arylesterase/paraoxonase activities of PON1. Furthermore, it was found that ox-LDL (P < 0.01) and lipid peroxides (P < 0.05) were significantly higher in men than in women. PON1 arylesterase activity was marginally affected by sex. The results of this study suggest that the anti-atherogenic properties of HDLs are not directly related to their total concentration and that PON1 activity determined towards synthetic compounds (paraoxon and phenyl acetate) reflects no association with markers of oxidative stress. Furthermore, it follows from our results that men are more susceptible to developing atherosclerosis compared to women.     

Clinical and laboratory aspects of a trichinellosis outbreak in Izmir, Turkey

Epidemiological, clinical and laboratory data were collected during an outbreak of trichinellosis, which occurred in Izmir, Turkey, between January and March 2004. The source of the infection was raw meatballs made with a mixture of uncooked beef and pork. Of 474 persons who were admitted at the Ataturk Training and Research Hospital during this period with a history of raw meatball consumption, the diagnosis of trichinellosis was confirmed for 154 (32.5%, 87 males and 67 females; mean age 31 years, range 6-67 years). Among persons with a confirmed diagnosis, 79% had myalgia, 77% weakness and malaise, 63% arthralgia, 40% jaw pain, 68% fever, 63% periorbital and/or facial oedema, 49% oedema at the trunk and limb, 42% abdominal pain, 40% nausea and vomiting, 28% diarrhoea, 23% subconjunctival haemorrhage, 25% macular or petechial rash, 4% subungual haemorrhage, 15% cardiac complaints and 0.2% neurological complaints. Nine patients (5.8%) were hospitalised due to severe myalgia (n = 2), high fever (n = 3), neurological manifestations (n = 1), thrombophlebitis (n = 2) and palmar erythema (n = 1). Eosinophilia was present in 88% of the confirmed cases at the admission. Elevated levels of serum creatine phosphokinase, lactic dehydrogenase and aspartate aminotransferase were detected in 72%, 70% and 16% of the confirmed cases, respectively. The seroconversion occurred in most of the infected people between the 4th and 6th weeks after the infection. All of the confirmed cases were treated with mebendazole. People with severe symptoms were treated also with prednisolone (60 mg/day for three days) and those with a moderately severe clinical pattern received a non-steroid anti-inflammatory drug (naproxen sodium, 550 mg/day). All confirmed cases recovered without any clinical sequela.     

New noncovalent inhibitors of penicillin-binding proteins from penicillin-resistant bacteria

Background

Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs β-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for β-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs.

Methodology/Principal Findings

Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains.

Conclusions

We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.     

The region of antithrombin interacting with full-length heparin chains outside the high-affinity pentasaccharide sequence extends to Lys136 but not to Lys139

The interaction of a well-defined pentasaccharide sequence of heparin with a specific binding site on antithrombin activates the inhibitor through a conformational change. This change increases the rate of antithrombin inhibition of factor Xa, whereas acceleration of thrombin inhibition requires binding of both inhibitor and proteinase to the same heparin chain. An extended heparin binding site of antithrombin outside the specific pentasaccharide site has been proposed to account for the higher affinity of the inhibitor for full-length heparin chains by interacting with saccharides adjacent to the pentasaccharide sequence. To resolve conflicting evidence regarding the roles of Lys136 and Lys139 in this extended site, we have mutated the two residues to Ala or Gln. Mutation of Lys136 decreased the antithrombin affinity for full-length heparin by at least 5-fold but minimally altered the affinity for the pentasaccharide. As a result, the full-length heparin and pentasaccharide affinities were comparable. The reduced affinity for full-length heparin was associated with the loss of one ionic interaction and was caused by both a lower overall association rate constant and a higher overall dissociation rate constant. In contrast, mutation of Lys139 affected neither full-length heparin nor pentasaccharide affinity. The rate constants for inhibition of thrombin and factor Xa by the complexes between antithrombin and full-length heparin or pentasaccharide were unaffected by both mutations, indicating that neither Lys136 nor Lys139 is involved in heparin activation of the inhibitor. Together, these results show that Lys136 forms part of the extended heparin binding site of antithrombin that participates in the binding of full-length heparin chains, whereas Lys139 is located outside this site.     

The effect of submergence on tuber production and dormancy in two subspecies ofBolboschoenus maritimus

Two subspecies ofBolboschoenus maritimus (L.)Palla were cultivated in submerged and non-submerged conditions. The production of tubers, the proportion of tubers remaining dormant and the time between production and reactivation were investigated. Total tuber production was found to be dependent on subspecies and may be considered a biological character of the subspecies.Bolboschoenus maritimus ssp.compactus, a subspecies of less, flooded habitats, produced more tubers with a greater capacity for growth leaving a smaller proportion of dormant tubers. Inactivity of tubers and their reactivation after one or two years were enhanced by submergence in both subspecies. The proportion of tubers that are permanently dormant as well as the dormancy of the whole plant are important features influencing the survival of populations ofB. maritimus over periods of unfavourable conditions.     

The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity.

From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two-chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X-ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha-carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha-carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy-terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this "occluding loop' provide positively charged anchors for the C-terminal carboxylate group of such polypeptide substrates. These structural features explain the well-known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2-side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo- and exopeptidase activity. The "occluding loop' does not allow cystatin-like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.     

Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation.

Three independent point mutations within residues 97 to 103 of the simian virus 40-small-t antigen (small-t) greatly reduced the ability of purified small-t to inhibit protein phosphatase 2A in vitro. These mutations affected the interaction of small-t antigen with the protein phosphatase 2A A subunit translated in vitro, and a peptide from the region identified by these mutations released the A subunit from immune complexes. When introduced into virus, the mutations eliminated the ability of small-t to enhance viral transformation of growth-arrested rat F111 cells. In contrast, the mutant small-t antigens were unimpaired in the transactivation of the adenovirus E2 promoter, an activity which was reduced by a double mutation in small-t residues 43 and 45.