Circulating oxidized low-density lipoprotein and common carotid artery intima-media thickness in a random sample of middle-aged men

Circulating oxidized low-density lipoprotein (oxLDL) has been suggested to play an important role in atherosclerosis development. According to previous observations, oxLDL correlates with clinically manifest coronary and carotid artery disease. We investigated the association between the oxLDL concentration measured directly in plasma and common carotid artery intima-media thickness (IMT) in a population-based, case-control study in middle-aged men from Southern Finland. oxLDL was determined in 214 men by a commercially available sandwich ELISA test (Mercodia). Carotid artery IMT was measured at 12 standardized segments by B-mode ultrasonography (at the near and far wall of the left and right common carotid arteries, bifurcations and internal carotid arteries), and the overall mean maximum IMT (MMaxIMT) was calculated. The MMaxIMT of the carotid arteries was significantly associated with circulating oxLDL (r_s=0.16, p=0.018). In a stepwise multiple regression model with MMaxIMT as dependent variable and systolic blood pressure, smoking, oxLDL, HDL cholesterol and apolipoprotein B as covariates, systolic blood pressure (=0.22, p<0.001), oxLDL (=0.15, p=0.022) and smoking (=0.17, p=0.014) showed an independent association with IMT (R²=0.10, p<0.001). Our results show that oxLDL measured directly from plasma is independently associated with subclinical carotid artery atherosclerosis in middle-aged men.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Arthritogenic properties of double-stranded (viral) RNA

Viral infections often lead to arthralgias and overt arthritic states. The inflammatogenic compound of the viruses giving rise to such an outcome has to date not been identified. Because expression of dsRNA is a common feature of all viruses, we decided to analyze whether this property leads to the induction of arthritis. Histological signs of arthritis were evident already on day 3 following intra-articular administration of dsRNA. Arthritis was characterized by infiltration of macrophages into synovial tissue. It was not dependent on acquired immune responses because SCID mice also raised joint inflammation. NF-kappa B was activated upon in vitro exposure to dsRNA, indicating its role in the induction/progression of arthritis. Importantly, we found that dsRNA arthritis was triggered through IL-1R signaling because mice being deficient for this molecule were unable to develop joint inflammation. Although dsRNA is typically recognized by Toll-like receptor 3, Toll-like receptor 3 knockout mice developed arthritis, indicating that some other receptors are instrumental in the inducing of inflammation. Our results from in vitro experiments indicate that proinflammatory cytokines and chemokines stimulating monocyte influx were readily triggered in response to stimulation with dsRNA. These findings demonstrate that viral dsRNA is clearly arthritogenic. Importantly, macrophages and their products play an important role in the development of arthritis triggered by dsRNA.     

Tracing of norovirus outbreak strains in mussels collected near sewage effluents

Noroviruses from mussels collected near sewage effluents were compared with local patient outbreak strains. Sequence analyses of RNA polymerase-capsid-poly(A)-3' (3.1-kilobase) regions confirmed the 99.9% similarity between genotype I.1 strains from mussels and patient strains from recreational-bathing outbreaks, indicating the potential usefulness of sentinel norovirus mussel studies in tracing human norovirus contamination of coastal waters.     

Is coproporphyrin III a copper-acquisition compound in Paracoccus denitrificans?

Paracoccus denitrificans is a soil bacterium which can respire aerobically and also denitrify if oxygen is absent. Both processes are highly dependent on copper enzymes and copper is therefore likely to be an essential trace element for the bacterium. If copper is not easily available, a copper-acquisition mechanism would be highly beneficial. In this paper, we have addressed the question of whether Paracoccus secretes a copper-acquisition compound functionally analogous to that found in some methanotrophs. Bacteria were grown both in copper-containing and copper-deficient denitrification media, cells were removed by centrifugation and the supernatant was analysed using chromatography and spectroscopy. Bacterial growth yield in the absence of copper was 70-80% of that in the copper-containing medium. A notable difference between the two culture conditions was that spent copper-deficient medium was pigmented, whereas the copper-containing medium was not. Spectrophotometry indicated that a red compound with an absorption maximum at 405 nm was produced under copper-limited conditions. In addition to the strong 405 nm maximum, the visible spectrum of the purified red molecule had weaker maxima at 535 nm and 570 nm, features typical of metallated tetrapyrroles. Mass spectrometry showed that the purified pigment had a molecular mass of 716.18. Moreover, the fine structure of the mass spectrum suggested the presence of zinc and was consistent with the chemical formula of C(36)H(36)N(4)O(8)Zn. The presence of zinc was also demonstrated using inductively coupled plasma atomic emission spectroscopy. Fragmentation analysis with mass spectrometry showed the release of consecutive 59 Da fragments, assignable to four -CH(2)-COOH moieties. Thin layer chromatography as well as NMR analysis of the C-13/N-15 labelled red pigment suggested that it is predominantly zinc coproporphyrin III with a minor fraction of metal-free coproporphyrin III. We propose that in a copper-poor environment P. denitrificans secretes coproporphyrin III for copper chelation and subsequent uptake of the bound copper into the cell. Consistent with this idea, cell yields of copper-deficient cultures grown in the presence of 1 microM copper-coproporphyrin III were 90-95% of the yields of cultures grown in the normal copper-containing media. Coproporphyrin III may work as a copper-acquisition compound in P. denitrificans.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

Vegan diet in physiological health promotion

We have performed a number of studies including dietary interventions and cross-sectional studies on subjects consuming uncooked vegan food called living food (LF) and clarified the changes in several parameters related to health risk factors. LF consists of germinated seeds, cereals, sprouts, vegetables, fruits, berries and nuts. Some items are fermented and contain a lot of lactobacilli. The diet is rich in fiber. It has very little sodium, and it contains no cholesterol. Food items like berries and wheat grass juice are rich in antioxidants such as carotenoids and flavonoids. The subjects eating living food show increased levels of carotenoids and vitamins C and E and lowered cholesterol concentration in their sera. Urinary excretion of sodium is only a fraction of the omnivorous controls. Also urinary output of phenol and p-cresol is lowered as are several fecal enzyme levels which are considered harmful. The rheumatoid arthritis patients eating the LF diet reported amelioration of their pain, swelling of joints and morning stiffness which all got worse after finishing LF diet. The composite indices of objective measures showed also improvement of the rheumatoid arthritis patients during the intervention. The fibromyalgic subjects eating LF lost weight compared to their omnivorous controls. The results on their joint stiffness and pain (visual analogue scale), on their quality of sleep, on health assessment questionnaire and on general health questionnaire all improved. It appears that the adoption of vegan diet exemplified by the living food leads to a lessening of several health risk factors to cardiovascular diseases and cancer. Rheumatoid patients subjectively benefited from the vegan diet which was also seen in serum parameters and fecal analyses.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).