No abstract

This study reports improvements in two of the key steps, lysis of indigenous cells and DNA purification, required for achieving a rapid nonselective protocol for extracting nucleic acids directly from sodium dodecyl sulfate (SDS)-treated sediment rich in organic matter. Incorporation of bead-mill homogenization into the DNA extraction procedure doubled the densitometrically determined DNA yield (11.8 jig of DNA* g [dry weight] of sediment-') relative to incorporation of three cycles of freezing and thawing (5.2 ,ug of DNA* g [dry weight] of sediment-'). The improved DNA extraction efficiency was attributed to increased cell lysis, measured by viable counts of sediment microorganisms which showed that 2 and 8%, respectively, survived the bead-mill homogenization and freeze-thaw procedures. Corresponding measurements of suspensions of viable Bacillus endospores demonstrated that 2 and 94% of the initial number survived. Conventional, laser scanning epifluorescence phase-contrast, and differential interference-contrast microscopy revealed that small coccoid bacterial cells (1.2 to 0.3 ,um long) were left intact after combined SDS and bead-mill homogenization of sediment samples. Estimates of the residual fraction of the fluorescently stained cell numbers indicated that 6% (2.2 X 108 cellsg [dry weight] of sediment-1) of the original population (3.8 X 109 cells * g [dry weight] of sediment-') remained after treatment with SDS and bead-mill homogenization. Thus, lysis of total cells was less efficient than that of cells which could be cultured. The extracted DNA was used to successfully amplify nahR, the regulatory gene for naphthalene catabolism in Pseudomonas putida G7, by PCR. By scaling down the mass of sediment extracted to 0.5 g and by using gel purification and SpinBind DNA purification cartridges, the time required to extract DNA from whole sediment samples was reduced to 2 h. Microbial ecologists, systematicists, and population geneticists have become increasingly interested in methods for complete, unbiased isolation of DNA (7, 9, 12, 16, 29, 30) and RNA (6, 8, 11, 19, 34, 36) from soils and sediments because such procedures promise to make the genomes of uncultured indigenous microorganisms available for molecular analysis. The ideal (2, 35, 36) is to circumvent the biases implicit in culture-based procedures by directly accessing the genes of naturally occurring microbial communities. But achieving this ideal requires overcoming a variety of interferences that diminish the quality, yield, and diversity of extracted nucleic acids. These interferences raise questions about the completeness of nucleic acid extraction, and about the representativeness of results based on the procedures. The popular direct lysis approach to DNA extraction and purification (24) may be dissected into the following conceptual steps: (i) washing the material to remove soluble components that may impair manipulation of subsequently isolated DNA; (ii) disruption of cells in the material to release DNA or RNA from the cells; (iii) sep...

Obstruction of a coronary artery or of any of its large branches has long been regarded as a serious accident. Several events contributed toward the prevalence of the view that this condition was almost always suddenly fatal. Parry's writings on angina pectoris and its relation to coronary disease, Jenner's observations on the same condition centering about John Hunter's case, Thorvaldsen's tragic death in the theater in Copenhagen with the finding of a plugged coronary, sharply attracted attention to the relation between the coronary and sudden death. In Germany Cohnheim supported the views of Hyrtl and Henle as to lack of considerable anastomosis, and as late as 1881 lent the influence of his name to the doctrine that the coronary arteries were end-arteries; his Leipsic necropsy experience, as well as experiments on dogs, forced him to conclude that the sudden occlusion of one of these vessels or of one of the larger branches, such as the ramus descendens of the left coronary, meant death within a few minutes. Other3

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Investigators have reported improved endurance performance and attenuated post-exercise muscle damage with carbohydrate-protein beverages (CHO+P) versus carbohydrate-only beverages (CHO). However, these benefits have been demonstrated only when CHO+P was administered in beverage-form, and exclusively in male subjects. Thus, the purposes of this study were to determine if an oral CHO+P gel improved endurance performance and post-exercise muscle damage compared to a CHO gel, and determine if responses were similar between genders. Thirteen cyclists (8 men, 5 women; VO(2)peak = 57.9 +/- 7.0 ml x kg(-1) x min(-1)) completed two timed cycle-trials to volitional exhaustion at 75% of VO(2)peak. At 15-minute intervals throughout these rides, subjects received CHO or CHO+P gels, which were matched for carbohydrate content (CHO = 0.15 g CHO x kg BW(-1); CHO+P = 0.15 g CHO + 0.038 g protein x kg BW(-1)). Trials were performed using a randomly counterbalanced, double-blind design. Subjects rode 13% longer (p < 0.05) when utilizing the CHO+P gel (116.6 +/- 28.5 minutes) versus the CHO gel (102.8 +/- 25.0 minutes). In addition, men (101.8 +/- 24.6; 114.8 +/- 26.2) and women (104.4 +/- 28.6; 119.6 +/- 34.9) responded similarly to the CHO and CHO+P trials, with no significant treatment-by-gender effect. Postexercise creatine kinease (CK) was not significantly different between treatments. However, CK increased significantly following exercise in the CHO trial (183 +/- 116; 267 +/- 214 U x L(-1)), but not the CHO+P trial (180 +/- 133; 222 +/- 141 U x L(-1)). Therefore, to prolong endurance performance and prevent increases in muscle damage, it is recommended that male and female cyclists consume CHO+P gels rather than CHO gels during and immediately following exercise.

Past research has shown that the most important areas for active sand movement in the northern part of the Chihuahuan Desert are mesquite-dominated desert ecosystems possessing sandy soil texture. The most active sand movement in the mesquite-dominated ecosystems has been shown to take place on elongated bare soil patches referred to as "streets". Aerodynamic properties of mesquite streets eroded by wind should be included in explaining how mesquite streets are more emissive sand sources than surrounding desert land. To understand the effects of wind properties, we measured them at two flat mesquite sites having highly similar soil textures but very different configurations of mesquite. The differences in wind properties at the two sites were caused by differences of size, orientation, and porosity of the mesquite, along with the presence of mesquite coppice dunes (sand dunes stabilized by mesquites growing in the dune and on its surface) found only at one of the two sites. Wind direction, u * (friction velocity), z 0 (aerodynamic roughness height) and D (zero plane displacement height) were estimated for 15-m tower and 3-m mast data. These aerodynamic data allowed us to distinguish five categories with differing potentials for sediment transport. Sediment transport for the five categories varied from unrestricted, free transport to virtually no transport caused by vegetation protection from wind forces. In addition, "steering" of winds below the level of the tops of mesquite bushes and coppice dunes allowed longer parallel wind durations and increased wind erosion for streets that aligned roughly SW-NE.

We report the amplification of bacterial genes from uninoculated surface and subsurface sediments by the polymerase chain reaction (PCR). PCR amplification of indigenous bacterial 16S ribosomal DNA genes was unsuccessful when subsurface sediment containing approximately 107 cells* g-1 was added directly to a PCR mixture. However, when 10 mg of sediment was inoculated with approximately 105 cells of Pseudononas putida G7, the nahAc naphthalene dioxygenase gene characteristic of the P. putida G7 NAH7 plasmid was detected by PCR amplification. Southern blotting of the PCR amplification product improved sensitivity to 103 to 104 cells from samples inoculated with P. putida G7, but controls with no sediment added showed that the PCR was partially inhibited by the sediments. Lysozyme-sodium dodecyl sulfate-freeze-thaw DNA extraction was combined with gel electrophoretic partial purification in the presence of polyvinylpyrrolidone to render DNA from indigenous bacteria in surface or subsurface sediment samples amplifiable by PCR using eubacterial 16S ribosomal DNA primers. The nahAc gene could also be amplified from indigenous bacteria by using nahAc-specific primers when PCR conditions were modified by increasing Taq and primer concentrations. Restriction digests of the nahAc amplification products from surface and subsurface sediments revealed polymorphism relative to P. putida G7. The procedures for DNA extraction, purification, and PCR amplification described here demonstrate that the PCR is a potentially useful tool in studies of functionand taxon-specific DNA from indigenous microbial communities in sediment and groundwater environments.