In vivo measurement of coronary circulation angiotensin-converting enzyme activity in humans

Angiotensin-converting enzyme (ACE) is present on the luminal surface of the coronary vessels, mostly on capillary endothelium. ACE is also expressed on coronary smooth muscle cells and on plaque lipid-laden macrophages. Excessive coronary circulation (CC)-ACE activity might be linked to plaque progression. Here we used the biologically inactive ACE substrate (3)H-labeled benzoyl-Phe-Ala-Pro ([(3)H]BPAP) to quantify CC-ACE activity in 10 patients by means of the indicator-dilution technique. The results were compared with atherosclerotic burden determined by coronary angiography. There was a wide range of CC-ACE activity as revealed by percent [(3)H]BPAP hydrolysis (30-74%). The atherosclerotic extent scores ranged from 0.0 to 66.97, and the plaque area scores ranged from 0 to 80 mm(2). CC-ACE activity per unit extracellular space (V(max)/K(m)V(i)), an index of metabolically active vascular surface area, was correlated with myocardial blood flow (r = 0.738; P = 0.03) but not with measures of the atherosclerotic burden. These results show that CC-ACE activity can be safely measured in humans and that it is a good marker of the vascular area of the perfused myocardium. It does not, however, reflect epicardial atherosclerotic burden, suggesting that local tissue ACE may be more important in plaque development.     

In-field labeling of western corn rootworm adults (Coleoptera: Chrysomelidae) with rubidium

Field and laboratory studies were conducted in 2000 and 2001 to determine the feasibility of mass marking western corn rootworm adults, Diabrotica virgifera virgifera LeConte, with RbCl in the field. Results showed that application of rubidium (Rb) in solution to both the soil (1 g Rb/plant) and whorl (1 g Rb/plant) of corn plants was optimal for labeling western corn rootworm adults during larval development. Development of larvae on Rb-enriched corn with this technique did not significantly influence adult dry weight or survival. Rb was also highly mobile in the plant. Application of Rb to both the soil and the whorl resulted in median Rb concentrations in the roots (5,860 ppm) that were 150-fold greater than concentrations in untreated roots (38 ppm) 5 wk after treatment. Additionally, at least 90% of the beetles that emerged during the first 3 wk were labeled above the baseline Rb concentration (5 ppm dry weight) determined from untreated beetles. Because emergence was 72% complete at this time, a significant proportion of the population had been labeled. Results from laboratory experiments showed that labeled beetles remained distinguishable from unlabeled beetles for up to 4 d postemergence. The ability to efficiently label large numbers of beetles under field conditions and for a defined period with virtually no disruption of the population provides an unparalleled opportunity to conduct mark-recapture experiments for quantifying the short-range, intrafield movement of adult corn rootworms.     

Microbial populations involved in cycling of dimethyl sulfide and methanethiol in freshwater sediments

Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related to Methanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene from M. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogen M. hollandica in freshwater sediments.     

Unnatural enantiomers of 5-azacytidine analogues: syntheses and enzymatic properties

2'-Deoxy-beta-L-5-azacytidine(L-Decitabine), beta-L-5-azacytidine, and derivatives were stereospecifically prepared starting from L-ribose or L-xylose. D- and L-enantiomers of 2'-deoxy-beta-5-azacytidine were weak substrates of human recombinant deoxycytidine kinase (dCK), whereas both enantiomers of beta-5-azacytidine or the L-xylo-analogues were not substrates of the enzyme. None of the reported derivatives of beta-L-5-azacytidine was a substrate of human recombinant cytidine deaminase (CDA).     

Synthesis and in vitro activity of D- and L-enantiomers of 5-(trifluoromethyl)uracil nucleoside derivatives

Recently, beta-L-nucleoside analogues have emerged as a new class of sugar modified nucleosides with potential antiviral and/or antitumoral activity. As a part of our ongoing research on this topic, we decided to synthesize 5-CF3-beta-L-dUrd (7), the hitherto unknown L-enantiomer of Trifluridine, an antiherpetic drug approved by FDA but only used in topical applications due to concomitant cytotoxicity. 5-CF3-beta-L-dUrd (7) as well as some other related L-nucleoside derivatives were stereospecifically prepared and tested in vitro against viral (HSV-1 and HSV-2) and human thymidine kinases (TK).     

Anti-HBV specific beta-L-2'-deoxynucleosides

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.     

Megagametophyte-derived linkage maps of white spruce ( Picea glauca) based on RAPD,SCAR and ESTP markers

We have constructed linkage maps for two parents of white spruce [ Picea glauca (Moench) Voss]. Haploid megagametophytes from 92 and 96 seeds of parents M2 and 80132, respectively, were analysed with RAPD, SCAR and ESTP markers. Fragments segregating in a 1:1 Mendelian ratio were classified and mapped using MAPMAKER, GMENDEL and JOINMAP. For M2, the analysis with JOINMAP resulted in 165 loci (152 RAPDs, 3 SCARs and 10 ESTPs) mapping to 23 linkage groups and covering 2,059.4 cM(Kosambi function, K). For 80132, the analysis resulted in 144 loci (137 RAPDs, 1 SCAR and 7 ESTPs) mapping to 19 linkage groups and covering 2,007.7 cM(K). The maps covered 87 and 73% of the entire genome of parents M2 and 80132, respectively. Similar results were obtained with MAPMAKER and GMENDEL. A comparison was made between the two individual maps and 16 loci were shared between the two maps.     

Characterization of low- and very-low-density hepatitis C virus RNA-containing particles

The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.     

A two-component modular approach for enhancing T-cell activation utilizing a unique anti-FcgammaRI-streptavidin construct and microspheres coated with biotinylated-antigen

The professional antigen presenting cell (APC) plays an essential role in the initiation and propagation of the acquired immune response. Thus, much work has been done in designing strategies that target vaccine antigen (Ag) to APC. Utilizing recombinant DNA technology, we have created a unique two-component system that delivers biotinylated Ag to the Fc gamma receptor type I (FcgammaRI) on APC. Our studies demonstrate that we can successfully engineer FcgammaRI-specific targeting element proteins that simultaneously bind both biotin and recognize FcgammaRI. Additionally, we are able to engineer biotinylated Ag, which form functional elements when adsorbed onto latex microspheres. Furthermore, the targeting and functional element components bind to each other and successfully form two-component immunogens. T-cell activation in response to targeted Ag-laden microspheres is 10- to 100-fold greater than the response to the non-targeted Ag-laden microspheres. This enhancement is 100- to 1000-fold greater than the responses generated to soluble Ag. Thus, our results suggest that specific targeting of Ag-laden microspheres to FcgammaRI may significantly enhance the adjuvant properties of microparticulate delivery systems. Further development of this system may help to elucidate the mechanisms involved in generating enhanced responses to APC-targeted vaccines and significantly advance vaccine technology.     

The effect of aluminium in Atlantic salmon (Salmo salar) with special emphasis on alkaline water

Atlantic salmon (Salmo salar) parr were exposed to aluminium under both steady state and non-steady state chemical conditions in alkaline water. Under alkaline (pH 9.5) steady state conditions, approximately 350 microg Al l(-1) (predominantly aluminate, Al(OH)(4)(-)) had no acute toxic effect on the salmon. The fish, however, showed a physiological response after 3 weeks of exposure ( approximately 300% increase in blood glucose concentration, about 30% increase in blood haematocrit, and about 15% decrease in plasma Cl(-) concentration). No increase in toxicity was evident under non-steady state conditions, i.e. lowering Al solubility as pH was lowered from 9.5 to 7.5. The results indicate that the toxicity of the aluminate ion (Al(OH)(4)(-)) is low, and particularly lower than the corresponding toxicity of cationic Al hydroxides. The effects observed in fish exposed to Al-rich water at pH 9.5 were counteracted as Al solubility was decreased by lowering pH to 7.5. This is contrary to previous observations where Al solubility has been lowered by increasing pH from 5.0 to 6.5.