Recombination-deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages

Mutations in the genes recA and recBC were constructed in the virulent Salmonella typhimurium strain 14028s. Both the recA and recBC mutants were attenuated in mice. The mutants were also sensitive to killing by macrophages in vitro. The recombination mutants were no longer macrophage sensitive in a variant line of J774 macrophage-like cells that fail to generate superoxide. This suggests that repair of DNA damage by Salmonella is necessary for full virulence in vivo and that the oxidative burst of phagocytes is one source of such DNA damage.     

Tissue distribution of bupivacaine enantiomers in sheep

rac-Bupivacaine HCl was infused intravenously to constant arterial blood drug concentrations in sheep using a regimen of 4 mg/min for 15 min followed by 1 mg/min to 24 h. At 24 h, arterial blood was sampled, the animal was killed with a bolus of KCl solution, then rapidly dissected and samples were obtained from heart, brain, lung, kidney, liver, muscle, fat, gut, and rumen. Tissue:blood distribution coefficients for (+)-(R)-bupivacaine exceeded those of (?)-(S)-bupivacaine (P < 0.05) for heart, brain, lung, fat, gut, and rumen by an overall mean of 43%. Blood:plasma distribution coefficients of (?)-(S)-bupivacaine exceeded those of (+)-(R)-bupivacaine by a mean of 29% and this offset the tissue:blood distribution coefficients so that the previously significant enantioselective differences disappeared. It is concluded that although enantioselectivity of bupivacame distribution is shown by the measured tissue:blood distribution coefficients, it is not shown when tissue:plasma water distribution coefficients are calculated, suggesting that there is no intrinsic difference between the bupivacaine enantiomers in tissue affinity. Sheep given fatal intravenous bolus doses of rac-bupivacaine had significantly greater concentrations of (+)-(R)-bupivacaine than (?)-(S)-bupivacaine in brain (P = 0.028) and ventricle (P = 0.036); these could augment the greater myocardial toxicity of this enantiomer found in vitro. © 1993 Wiley-Liss, Inc.     

MITOCHONDRIAL-DNA ANALYSES AND THE ORIGIN AND RELATIVE AGE OF PARTHENOGENETIC LIZARDS (GENUS CNEMIDOPHORUS). III. C. VELOX AND C. EXSANGUIS

Mitochondrial DNAs (mtDNAs) of two unisexual, parthenogenetically reproducing species of whiptail lizards (Cnemidophorus velox and C. exsanguis) and their bisexual relatives were compared by restriction-enzyme analysis to assess levels of mtDNA variation and to establish the maternal ancestry of the unisexuals. No cleavage-site differences were found to be diagnostic between C. velox and C. exsanguis mtDNAs, suggesting an ancestry rooted in the same maternal lineage. The mtDNA of the unisexuals is relatively homogeneous, indicating that these lineages are of recent origin. Phylogenetic analysis revealed that the maternal ancestor of both C. velox and C. exsanguis was most probably C. burti stictogrammus, C. costatus barrancorum, or an unidentified taxon closely related to them. In addition, the mtDNA analyses demonstrate conclusively that the triploid species C. velox could not have been formed by the fertilization of an unreduced (diploid) C. inornatus egg, further strengthening the hypothesis that parthenogenesis in Cnemidophorus results from hybridization.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.     

VASOMOTOR RESPONSES OF ISOLATED PERIPHERAL BLOOD VESSELS FROM BOWHEAD WHALES: THERMOREGULATORY IMPLICATIONS

Temperature regulation in bowhead whales, Balaena mysticetus , is supported by the characteristic cetacean peripheral circulation, especially notable in the tail flukes. Blood vessels serving this function consist of countercurrent heat exchangers (network of veins surrounding a central artery) favoring heat conservation and an alternate routing via arteriovenous anastomoses (AVAs) providing for heat dissipation. We tested the vasomotor responses of isolated segments of countercurrent arteries and AVAs from the bowhead tail flukes to norepinephrine (NOR), the sympathetic adrenergic neurotransmitter. Isometric tension developed during exposure to a micromolar concentration of NOR was consistently higher in AVAs than in arteries. Accordingly, the AVAs are subject to sympathetic vasoconstriction, and this activation directs blood flow to countercurrent heat exchangers and results in heat conservation. In contrast, AVA relaxation by reduced sympathetic activation favors increased blood flow through AVAs and consequent peripheral heat loss.     

A Comparison of Remote Sensing and Ground-Based Methods for Monitoring Wetland Restoration Success

Abstract Efficient and accurate vegetation sampling techniques are essential for the assessment of wetland restoration success. Remotely acquired data, used extensively in many locations, have not been widely used to monitor restored wetlands. We compared three different vegetation sampling techniques to determine the accuracy associated with each method when used to determine species composition and cover in restored Pacific coast wetlands dominated by Salicornia virginica (perennial pickleweed). Two ground‐based techniques, using quadrat and line intercept sampling, and a remote sensing technique, using low altitude, high resolution, color and color infrared photographs, were applied to estimate cover in three small restoration sites. The remote technique provided an accurate and efficient means of sampling vegetation cover, but individual species could not be identified, precluding estimates of species density and distribution. Aerial photography was determined to be an effective tool for vegetation monitoring of simple (i.e., single‐species) habitat types or when species identities are not important (e.g., when vegetation is developing on a new restoration site). The efficiency associated with these vegetation sampling techniques was dependent on the scale of the assessment, with aerial photography more efficient than ground‐based sampling methods for assessing large areas. However, the inability of aerial photography to identify individual species, especially mixed‐species stands common in southern California salt marshes, limits its usefulness for monitoring restoration success. A combination of aerial photography and ground‐based methods may be the most effective means of monitoring the success of large wetland restoration projects.     

Characterization of cpsF and its product CMP-N-acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli

Group B Streptococcus (GBS) is the foremost cause of neonatal sepsis and meningitis in the United States. A major virulence factor for GBS is its capsular polysaccharide, a high molecular weight polymer of branched oligosaccharide subunits. N -acetylneuraminic acid (Neu5Ac or sialic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP-Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient in CMP-Neu5Ac-synthetase activity (Wessels et al ., 1992). In this paper, we report the identification and characterization of cpsF , a gene interrupted by transposon insertion in the previously described Neu5Ac-deficient mutant. The predicted amino acid sequence of the cpsF gene product shares 57% similarity and 37% identity with CMP-Neu5Ac synthetase encoded by the Escherichia coli K1 gene, neuA . The enzymatic function of the protein encoded by cpsF was established by cloning the gene in E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the condensation of CTP with Neu5Ac to form CMP-Neu5Ac. In addition, when a CMP-Neu5Ac synthetase-deficient mutant of E. coli K1 was transformed with cpsF , K1 antigen expression was restored. We conclude that cpsF encodes CMP-Neu5Ac synthetase in type III GBS, and that the GBS enzyme can function in the capsule-synthesis of a heterologous bacterial species.     

Managing the pattern of forest harvest: lessons from wildfire

Managing forests for sustainable use requires that both the biological diversity of the forests and a viable forest industry be maintained. A current approach towards maintaining biological diversity is to pattern forest management practices after those of natural disturbance events. This paradigm hypothesizes that ecological processes will be maintained best where active management approximates natural disturbance events. The forest management model now used in most sub-boreal and boreal forests calls for regularly dispersed clearcuts no greater than 60–100 ha in size. However, the spatial characteristics of the landscape produced by this model are distinctly different from the historic pattern generated by wildfire, which was heretofore the dominant stand-replacing process in these forests. Wildfire creates a more complex landscape spatial pattern with greater range in patch size and more irregular disturbance boundaries. Individual wildfires are often over 500 ha but leave patches of unburned forest within them. The combination of these attributes is not present in recent clearcuts. Allowing a proportion of larger (i.e.>500ha) harvest units may provide distinct economic advantages that could outweight the opportunity costs of leaving some patches of forest behind. For the forest type examined, further evaluation of modelling forest harvest patterns more closely after the patterns created by wildfire is required as it may achieve a good balance and strike a suitable compromise between certain ecological and economic objectives of sustainable development.     

Evaluation of a remotely operated vehicle (ROV) as a tool for studying the distribution and abundance of zooplankton

Conventional methods for measuring zooplankton distributionsare too laborious and time consuming to permit sufficient temporaland spatial resolution in many instances. An ability to makemore efficient and precise measurements would be useful. Weevaluated the potential for using the video system of a commerciallyavailable remotely operated vehicle (ROV) to measure the distributionand abundance of zooplankton by calibrating ROV counts withcounts based on a conventional sampling procedure (a Schindlertrap), and by using an ROV to measure the density of zooplanktonin a small lake. As configured here, this particular ROV wassuitable for measuring the density of the cladocerans Daphniaand Holopedium. It was also suitable for assessing the distribution,but not absolute densities, of Chaoborus and Leptodora. Imagequality was inadequate for quantitative estimates of copepod(Diaptomus minutus) abundance, and prevented us from studyingbehavioral responses of copepods to the vehicle. We concludethat the ROV has at least three useful features: it can be usedto locate patches of those species that are imaged effectively;a large number of samples (videotapes) can be collected almostsynoptically with high spatial resolution; the ROV enables insitu observation of zooplankton. The ROV also has three importantlimitations: the small image volume makes it difficult to studyrare organisms; inadequate image resolution precludes studiesof relatively small organisms (e.g. the calanoid copepod D.minutus);zooplankton respond to the presence of the ROV.     

Nutrient uptake relationship to root characteristics of rice

Data on root parameters and distribution are important for an improved understanding of the factors influencing nutrient uptake by a crop. Therefore, a study was conducted on a Crowley silt loam at the Rice Research and Extension Center near Stuttgart, Arkansas to measure root growth and N, P and K uptake by three rice (Oryza sativa L.) cultivars at active tillering (36 days after emergence (DAE)), maximum tillering (41 DAE), 1.25 cm internode elongation (55 DAE), booting (77 DAE) and heading (88 DAE). Soil-root core samples were taken to a depth of 40 cm after plant samples were removed, sectioned into 5 cm intervals, roots were washed from soil and root lengths, dry weights and radii were measured. Root parameters were significantly affected by the soil depth × growth stage interaction. In addition, only root radius was affected by cultivar. At the 0- to 5-cm soil depth, root length density ranged from 38 to 93 cm cm^-3 throughout the growing season and decreased with depth to about 2 cm cm^-3 in the 35- to 40-cm depth increment. The increase in root length measured with each succeeding growth stage in each soil horizon also resulted in increased root surface area, hence providing more exposed area for nutrient uptake. About 90% of the total root length was found in the 0- to 20-cm soil depth throughout the season. Average root radius measured in the 0- to 5-cm and 35- to 40-cm depth increments ranged from 0.012 to 0.013 cm and 0.004 to 0.005 cm, respectively throughout the season. Total nutrient uptake by rice differed among cultivars only during vegetative growth. Differences in total nutrient uptake among the cultivars in the field appear to be related to absorption kinetics of the cultivars measured in a growth chamber study. Published with permission of the Arkansas Agricultural Experiment Station.