Effects of an experimental acid pulse on invertebrates in a high altitude Sierra Nevada stream

The effects of pulsed acidification on invertebrate densities and drift, and water chemistry, in a high altitude Sierra Nevada stream were measured using artificial stream channels. Water was diverted from the Marble Fork of the Kaweah River, California, U.S.A., through twelve replicate channels; however, low flow in the summer of 1985 eliminated all but four of these channels. Channels were stocked with natural substrates and organisms from the Marble Fork of the Kaweah River. After a three week acclimation period, we simulated a low pH rain event by adding acid (H₂SO₄ and HNO₃) to two of the channels, reducing pH to 5.0 for 6 hours. The other two channels acted as controls (pH 6.4). During acid additions, Baetis spp. drift in acidified channels was ca. 7 times higher than in control channels (F = 39.02, p < 0.025; data fourth root transformed, ANOVA), and the percentage of drifting baetids that was dead was significantly higher in acidified than control channels (46% vs. 0%, F = 29.86, p < 0.05; arcsine square root transformed data, ANOVA). Other taxa showed no significant drift responses, and benthic densities of all taxa showed no effects two days after acidification, probably owing to rapid recolonization by invertebrate drift in influent waters. Stream chemistry data are presented; heavy metal concentrations did not significantly increase in the 2 m stream channels.     

Influence of the biomechanical environment on the femoral stem insertion and vibrational behavior: a 3-D finite element study

Biomechanics and Modeling in Mechanobiology - The long-term success of cementless surgery strongly depends on the implant primary stability. The femoral stem initial fixation relies on multiple...     

Substrate and inhibitor specificity of 3-hydroxy-3-methylglutaryl-CoA reductase determined with substrate-analogue CoA-thioesters and CoA-thioethers.

1) Analogues of 3-hydroxy-3-methylglutaryl-CoA were prepared in which the substituents at C-3 of the acyl residue were altered. The same analogues were additionally modified by replacement of the thioester oxygen by hydrogen to yield reduction-resistant CoA-thioethers. The interaction of both types of CoA derivatives with a 58-kDa catalytic fragment of human 3-hydroxy-3-methylglutaryl-CoA reductase was studied. 2) This enzyme reduces glutaryl-CoA at a very low rate whereas 3-hydroxyglutaryl-CoA is well reduced, the maximal rate of reduction being 7% that of the physiological substrate. Only half of total 3-hydroxyglutaryl-CoA was attacked, thus reflecting the stereo-specificity of the enzyme for (3S)-3-hydroxy-3-methylglutaryl-CoA. The results invalidate the hitherto assumed absolute substrate specificity of the enzyme. 3) The affinity of both 3-hydroxyglutaryl-CoA and its thioether variant S-(4-carboxy-3-hydroxybutyl)CoA to the reductase, Ki = 0.3 microM and Ki = 0.4 microM, respectively, is higher than that of the physiological substrate, Km = 1.5 microM (data related to (S)-diastereomer). The results show for the first time that the methyl-group effect observed with the inhibitor lovastatin is an intrinsic property of the enzyme. 4) All of the prepared CoA derivatives are purely competitive inhibitors of the reductase, the affinities varying within a range of two powers of ten (Ki = 0.3-32 microM). On variation of the substituents at C-3 of the acyl residue of the physiological substrate the affinity of both CoA-thioesters and CoA-thioethers increases in the sequence CH2, C(CH3)2, CH(CH3), C(OH)CH3, CH(OH).     

Identification of two distinct microtubule binding domains on recombinant rat MAP 1B.

A set of partially overlapping cDNA clones covering 9 kb of continuous sequence encoding the high molecular weight microtubule-associated protein (MAP) 1B, was isolated from a rat brain library in lambda gt11. The protein encoded was immunoreactive with monoclonal antibodies raised against calf MAP 1B, rat MAP 1X, and rat MAP 5, as shown by immunoblotting. Using Northern blot analysis, it was shown that the level of MAP 1B mRNA increased dramatically upon nerve growth factor-induced PC12 cell differentiation. The expression of polypeptides encoded by cDNA constructs, in conjunction with microtubule binding assays, revealed two separate microtubule binding domains, corresponding to sequences at the 5' and 3' end of the mRNA. As shown by DNA sequencing, the binding domain encoded by 5' terminal sequences consisted of the basic repeat motif KKEE(I/V), previously identified in mouse MAP 1B (Noble, M., S. A. Lewis, N. J. Cowan, J. Cell Biol. 109, 3367-3376 (1989)). The second binding domain, too, was found to be basic, but without any apparent repeat structure. It is concluded that single proteolytically unprocessed MAP 1B molecules would have the potential to function as microtubule cross-linkers.     

Characterization of Tn2411 and Tn2410, two transposons derived from R-plasmid R1767 and related to Tn2603 and Tn21

Two transposable elements, Tn2410 and Tn2411, were isolated from Salmonella typhimurium R-factor R1767. They have sizes of 18.5 and 18.0 kilobases, respectively. Tn2411 mediates resistance to streptomycin, sulfonamides, and mercury. In Tn2410, the streptomycin resistance gene was replaced by a gene coding for the production of the beta-lactamase OXA-2, which is responsible for ampicillin resistance. Physical and functional maps of both transposons were compared with those of Tn21, Tn4, and Tn2603. From these data it appeared that Tn21 could be an ancestral transposon from which Tn2411, Tn2410, Tn2603, and Tn4 were evolved by the addition or deletion of small DNA segments.     

Development of autotaxin inhibitors: A series of zinc binding triazoles

A series of inhibitors of Autotaxin (ATX) has been developed using the binding mode of known inhibitor, PF-8380, as a template. Replacement of the benzoxazolone with a triazole zinc-binding motif reduced crystallinity and improved solubility relative to PF-8380. Modification of the linker region removed hERG activity and led to compound 12 – a selective, high affinity, orally-bioavailable inhibitor of ATX. Compound 12 concentration-dependently inhibits autotaxin and formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic experiments.     

Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

This study examines the role of L-selectin in monocyte adhesion to arterial endothelium, a key pathogenic event of atherosclerosis. Using a nonstatic (rotation) adhesion assay, we observed that monocyte binding to bovine aortic endothelium at 4°C increased four to nine times upon endothelium activation with tumor necrosis factor (TNF)-α. mAb-blocking experiments demonstrated that L-selectin mediates a major part (64 ± 18%) of monocyte attachment. Videomicroscopy experiments performed under flow indicated that monocytes abruptly halted on 8-h TNF-α–activated aortic endothelium, ~80% of monocyte attachment being mediated by L-selectin. Flow cytometric studies with a L-selectin/IgM heavy chain chimeric protein showed calcium-dependent L-selectin binding to cytokine-activated and, unexpectedly, unactivated aortic cells. Soluble L-selectin binding was completely inhibited by anti–L-selectin mAb or by aortic cell exposure to trypsin. Experiments with cycloheximide, chlorate, or neuraminidase showed that protein synthesis and sulfate groups, but not sialic acid residues, were essential for L-selectin counterreceptor function. Moreover, heparin lyases partially inhibited soluble L-selectin binding to cytokine-activated aortic cells, whereas a stronger inhibition was seen with unstimulated endothelial cells, suggesting that cytokine activation could induce the expression of additional ligand(s) for L-selectin, distinct from heparan sulfate proteoglycans. Under flow, endothelial cell treatment with heparinase inhibited by ~80% monocyte attachment to TNF-α–activated aortic endothelium, indicating a major role for heparan sulfate proteoglycans in monocyte–endothelial interactions. Thus, L-selectin mediates monocyte attachment to activated aortic endothelium, and heparan sulfate proteoglycans serve as arterial ligands for monocyte L-selectin.     

Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCR

The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10⁵ to 10¹⁰ CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10⁵ CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.     

Regional coherence of climatic and lake thermal variables of four lake districts in the Upper Great Lakes Region of North America

1. Within a region with common climatic conditions, lake thermal variables should exhibit coherent variability patterns to the extent to which they are not influenced by lake specific features such as morphometry and water clarity. We tested the degree of temporal coherence in interannual variability for climatic variables (air temperature and solar radiation) among four lake districts in the Upper Great Lakes Region. We also tested the degree of coherence of lake thermal variables (near‐surface temperature, eplimnetic temperature, hypolimnetic temperature and thermocline depth) for lakes within these districts. 2. Our four lake districts included the Experimental Lakes Area in north‐western Ontario, the Dorset Research Centre area north of Toronto, Ontario, the Northern Highland Lake District in northern Wisconsin, and the Yahara Lakes near Madison in southern Wisconsin. Seventeen lakes were analyzed for lake thermal variables dependent on stratification. Another five lakes were added for the analysis of near‐surface temperature. 3. The analysis tested whether for monthly and summer means, the climate (air temperature and solar radiation) across the four lake districts was coherent interannually and whether variables which measure the thermal structure of the lakes were coherent interannually among lakes within each lake district and across the four lake districts. 4. Temporal coherence was estimated by the correlation between lake districts for meteorological variables and between lake pairs for lake thermal variables. Mean coherence and the percentage of correlations exceeding the 5% significance level were derived both within and between lake districts for lake thermal variables. 5. Across the four lake districts, summer mean air temperature was highly coherent while summer solar radiation was less coherent. Approximately 60–80% of the interannual variation in mean summer air temperature at a site occurred across the entire region. Less than 45% of the variation in solar radiation occurred across sites. 6. Epilimnetic temperature and the near‐surface temperature were highly coherent both within and between lake districts. The coherence of thermocline depth within and between lake districts was weaker. Hypolimnetic temperature was not coherent between lake districts for most lake pairs. It was coherent among lakes within some lake districts. 7. The influences of local weather and differences among lakes in water clarity are discussed in the context of differences in levels of coherence among lake thermal variables and among lake pairs for a given variable.