Comparative actions of salicylate on the amphibian lateral line and guinea pig cochlea

1. Salicylate actions on afferent nerve activity in the Xenopus lateral line and on cochlear potentials in guinea pig were investigated. 2. In the lateral line, salicylate (0.3-2.5 mM) suppressed spontaneous activity, water motion evoked excitation and responses to L-glutamate (1-2 mM) and kainate (10-20 microM). 3. In the guinea pig, salicylate (0.6-10 mM) suppressed the compound action potential (CAP) and increased N1 latency at low but not high sound intensities. 4. In the lateral line salicylate action may involve an antagonism of the hair-cell transmitter on the afferent nerve. 5. In the cochlea salicylate may suppress the active process or cochlear amplifier.     

Indexes to the reassociation and stability of solution DNA hybrids

Summary The computation, assumptions, and properties of DNA-hybrid stability and reassociation indexes were reviewed. Different methods of computing the same index typically yielded similar values. However, because dissociation curves change from asymmetric to symmetric as increasingly divergent DNAs are compared, adequate determination of mode required fitting a complex function. Delta Tm, delta mode, and delta T50H correlated well up to ca. 12, and all were found to be useful indexes of genomic similarity in that range. They also exhibited similar levels of error, even though T50H comprises a percent reassociation component with relatively large variance. At greater distances, the delta Tm scale became markedly compressed because of the boundary imposed by the temperature of hybrid formation (incubation temperature). Though not compressed or technically limited by it, delta mode and delta T50H could not be extrapolated with certainty below the incubation temperature. Among theoretical problems discussed: Tm and mode index an increasingly small percentage of the genome as the extent of reassociation decreases, and they may compare different genomic segments as DNAs become highly diverged. T50H relies upon the assumptions that all sequences evolve at a constant rate and that reassociation behavior is the same among all sequences regardless of their extent of divergence. Tm and T50H may be biased by selfhybridization of repetitive elements or cross-hybridization of paralogous sequences. Delta mode is free of such biases as long as the genomes under comparison are not too diverged. No index was found to be best in all circumstances.     

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Mycorrhizal effects on potassium fluxes by northwest coniferous seedlings

In ectomycorrhizae, the relative abilities of mycobiont and host plant to take up and store inorganic nutrients are not easily determined due to the intimate physical relationship of the two components forming the association. Since compartmental analysis of solute elution can estimate cellular compartment pool sizes and unidirectional fluxes across membranes, we have used this method to study ectomycorrhizal coniferous roots. Rubidium-86, used as a tracer for potassium, was loaded into and eluted from intact roots of nonmycorrhizal and mycorrhizal (with the fungus Hebeloma crustuliniformme [Bull.: St. Amans Quél] Douglas fir (Pseudotsuga menziesii [Mirb.] Franco), western hemlock (Tsuga heterophylla [Raf.] Sarg.) and Sitka spruce (Picea sitchensis [Bong.] Carr.) seedlings.

Mycorrhizas significantly increased ⁸⁶Rb uptake rates while decreasing the amount of ⁸⁶Rb released to the external solution. Using compartmental analysis, the flux data suggest that the primary mycorrhizal effects were to increase inward potassium fluxes across the fungal tonoplast and to decrease potassium efflux across the fungal tonoplast, as compared with nonmycorrhizal seedling roots. The result was greater potassium storage, presumably in the fungal vacuole. The three coniferous species responded differently to fungal infection with respect to potassium fluxes. Both cytoplasmic and vacuolar fluxes for mycorrhizal hemlock were 2-fold greater than for spruce and 3-fold greater than for Douglas fir. These results demonstrate the usefulness of compartmental analysis for study of ion fluxes in intact mycorrhizal root systems and suggest that the fungal tonoplast may be the site for regulation of potassium fluxes in these coniferous roots.

     

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

A nitrogen fertilization field study of carbon-13 and nitrogen-15 transfers in ectomycorrhizas of Pinus sabiniana

Ectomycorrhizal (EM) fungi form relationships with higher plants; plants transfer C to fungi, and fungi transfer nutrients to their host. While evidence indicates that this interaction is largely mutualistic, less is known about how nutrient supply and EM associates may alter C and nutrient exchanges, especially in intact plant-soil-microbe systems in the field. In a dual-labeling experiment with N fertilization, we used C and N stable isotopes to examine in situ transfers in EM pine trees in a Pinus sabiniana woodland in northern California. We added ¹⁵NH₄SO₂ and ¹³CO₂ to track ¹³C transfer from pine needles to EM roots and ¹⁵N transfer from soil to EM roots and pine needles. Transfers of ¹³C and ¹⁵N differed with EM morphotype and with N fertilization. The brown morphotype received the least C per unit of N transferred (5:1); in contrast red and gold morphotypes gained more C and transferred less N (17:1 and 25:1, respectively). N fertilization increased N retention by ectomycorrhizas (EMs) but did not increase N transfer from EMs to pine needles. Therefore N fertilization positively affected both nutrient and C gains by EMs, increasing net C flows and N retention in EMs. Our work on intact and native trees/EM associations thereby extends earlier conclusions based on pot studies with young plants and culturable EM fungi; our results support the concept that EM-host relationships depend on species-level differences as well as responses to soil resources such as N.     

Identification of a major heparin-binding site in kallistatin

Kallistatin is a heparin-binding serine proteinase inhibitor (serpin), which specifically inhibits human tissue kallikrein by forming a covalent complex. The inhibitory activity of kallistatin is blocked upon its binding to heparin. In this study we attempted to locate the heparin-binding site of kallistatin using synthetic peptides derived from its surface regions and by site-directed mutagenesis of basic residues in these surface regions. Two synthetic peptides, containing clusters of positive-charged residues, one derived from the F helix and the other from the region encompassing the H helix and C2 sheet of kallistatin, were used to assess their heparin binding activity. Competition assay analysis showed that the peptide derived from the H helix and C2 sheet displayed higher and specific heparin binding activity. The basic residues in both regions were substituted to generate three kallistatin double mutants K187A/K188A (mutations in the F helix) and K307A/R308A and K312A/K313A (mutations in the region between the H helix and C2 sheet), using a kallistatin P1Arg variant as a scaffold. Analysis of these mutants by heparin-affinity chromatography showed that the heparin binding capacity of the variant K187A/K188A was not altered, whereas the binding capacity of K307A/R308A and K312A/K313A mutants was markedly reduced. Titration analysis with heparin showed that the K312A/K313A mutant has the highest dissociation constant. Like kallistatin, the binding activity of K187A/K188A to tissue kallikrein was blocked by heparin, whereas K307A/R308A and K312A/K313A retained significant binding and inhibitory activities in the presence of heparin. These results indicate that the basic residues, particularly Lys(312)-Lys(313), in the region between the H helix and C2 sheet of kallistatin, comprise a major heparin-binding site responsible for its heparin-suppressed tissue kallikrein binding.