Estimate of mean tissue O2 consumption at onset of exercise in males

A mathematical model has been developed that permitted the calculation of the flow-weighted mean tissue O2 consumption (VO2T) at the onset of a step increase in work rate. From breath-by-breath measurements of alveolar O2 consumption (VO2A) and cardiac output (Q) by impedance cardiography and assumptions about the site of depletion of O2 stores, the rate of change in O2 stores (VO2s) was determined. The sum of VO2A + VO2s = VO2T. Six very fit males performed six repetitions of each of two step increases in work rate. STlo was a transition from rest to 100-W cycling; SThi was a transition from 100- to 200-W cycling. For each work rate transition, the responses of VO2A and Q were averaged over the six repetitions of each subject and the model was solved to yield VO2T. The responses of VO2A, VO2T, and Q after the increase in work rate were fit with a monoexponential function. This function included a time constant and time delay, the sum of which gave the mean response time (MRT). In the STlo test, the MRT of VO2A (24.9 +/- 1.1 s, mean +/- SE) was longer than that of VO2T (15.3 +/- 1.3 s) and of Q (16.5 +/- 6.5 s) (P less than 0.05). The MRT of VO2T and Q did not differ significantly. Also for SThi, the MRT of VO2A (34.4 +/- 3.3 s) was significantly longer than that of VO2T (30.0 +/- 3.4 s) (P less than 0.05). The MRT of VO2T and Q (30.3 +/- 5.5 s) were not significantly different at this work rate either.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of sequential immune complexes in infective endocarditis by Western blot analysis

A patient with cutaneous vasculitis during infective endocarditis due to Lactobacillus casei was studied. Immune complexes (IC) were isolated from serum at the time of diagnosis and after 4 wk of therapy. Purification of IC used differential polyethylene glycol precipitation and competitive binding to staphylococcal protein A. In situ radioiodination of IC was performed, followed by SDS-polyacrylamide gel electrophoresis (PAGE). Anti-IC antisera were raised in rabbits by immunization with purified IC. IC were characterized by SDS-PAGE followed by electrophoretic transfer to nitrocellulose, incubation with antiserum and then with 125I protein A, and autoradiography. Although early and late IC differed quantitatively, there were no differentiating immunochemical features. Both IC contained a 60,000 dalton component that did not react with preimmune serum nor with anti-normal human serum. This component reacted with antiserum rendered specific for L. casei by affinity chromatography. The restricted antigen-antibody representation in IC contrasted with a wider panel of antibody activity in patient serum. The Western blot analysis proves to be an ideal method for the characterization of IC because of its sensitivity, dissociative capability, and preservation of immunoreactivity. IC isolated at a time removed from the original antigenic challenge may provide insight into the nature of the inciting antigen.     

Climatic sensitivity of species’ vegetative and reproductive phenology in a Hawaiian montane wet forest

Understanding how tropical tree phenology (i.e., the timing and amount of seed and leaf production) responds to climate is vital for predicting how climate change may alter ecological functioning of tropical forests. We examined the effects of temperature, rainfall, and photosynthetically active radiation (PAR) on seed phenology of four dominant species and community-level leaf phenology in a montane wet forest on the island of Hawaiʻi using monthly data collected over ~ 6 years. We expected that species phenologies would be better explained by variation in temperature and PAR than rainfall because rainfall at this site is not limiting. The best-fit model for all four species included temperature, rainfall, and PAR. For three species, including two foundational species of Hawaiian forests (Acacia koa and Metrosideros polymorpha), seed production declined with increasing maximum temperatures and increased with rainfall. Relationships with PAR were the most variable across all four species. Community-level leaf litterfall decreased with minimum temperatures, increased with rainfall, and showed a peak at PAR of ~ 400 μmol/m²s⁻¹. There was considerable variation in monthly seed and leaf production not explained by climatic factors, and there was some evidence for a mediating effect of daylength. Thus, the impact of future climate change on this forest will depend on how climate change interacts with other factors such as daylength, biotic, and/or evolutionary constraints. Our results nonetheless provide insight into how climate change may affect different species in unique ways with potential consequences for shifts in species distributions and community composition.     

Electron microscopic identification of supercoiled regions in complex DNA structures

When intracellular lambda replicative intermediates (theta structures) are intercalated with psoralen and then irradiated with long wavelength ultraviolet light (u.v.), interstrand crosslinks are produced. After purification and denaturation of these theta structures, a global difference in denaturation can be observed by electron microscopy; parental sections are essentially native whereas daughter segments are highly denatured. This difference can be explained if parental sections are covalently continuous (and therefore able to supercoil) and daughter segments are not. Due to the higher thermal stability of supercoiled DNA, parental DNA will remain native while daughter sections will denature. Because these structures are crosslinked, the thermal treatment does not lead to dissociation of the highly denatured daughter strands. Experiments with simple negatively supercoiled plasmid circles support the above conclusions. When circles are crosslinked with psoralen-u.v. and then denatured, they remain native because of the higher thermal stability of covalently closed structures. If the circles are linearized before heating but after the psoralen-u.v. treatment, the thermal stability effect is eliminated and the molecules become highly denatured. In this case, however, the crosslinking density is found to be higher than in samples linearized before psoralen-u.v. treatment. This, therefore, shows that crosslinking density also reflects the superhelical state of the molecule at the time of psoralen-u.v. treatment. Two different properties can be used to discriminate between supercoiled and covalently discontinuous domains in complex DNA structures. First, supercoiled regions remain native while covalently discontinuous segments denature following a thermal treatment. This effect requires that covalent continuity exists up to and during the heating treatment. Second, because negative superhelicity enhances psoralen intercalation, crosslinking density is higher in these regions. Even if supercoiled domains are destroyed after the psoralen-u.v. treatment, the imprint of superhelicity is retained and can be recognized as a higher than normal crosslinking density.     

Characterization of Holliday structures in FLP protein-promoted site-specific recombination.

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cell. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C.     

Modulation of the mitogenic response of an epidermal growth factor-dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor-beta

The stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGR receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up-regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high-affinity receptors (less than 500) are required for EGF-dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF-stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin-like growth factors I or II (IGF-I, IGF-II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF-I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor-beta (TGF-beta) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF-beta receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF-beta) significantly altered 125I-EGF binding characteristics in MK cells, suggesting a point of action distal to 125I-EGF binding.     

Experimental formulations ofBacillus sphaericus for the control of anopheline and culicine larvae

Summary Four experimental formulations ofBacillus sphaericus Neide (2362 isolate) were evaluated for larvicidal activity against culicine and anopheline larvae in several natural and artificial habitats. A granular formulation (5% primary powder) was tested against natural populations of mosquitoes in two simulated habitats in Florida and in maturing and reflooded rice fields in Louisiana. Larvae ofCulex quinquefasciatus Say were reduced by 97 and 99% after application of the granules at the rate of 10 kg/ha to polluted tanks and 2.5 kg/ha to sod-lined potholes, respectively. Anopheline andPsorophora columbiae (Dyar and Knab) larvae were reduced by 68 and 92–100%, respectively, after application of 5 kg granules/ha to rice fields. A flowable concentrate (12.8% primary powder) applied to unpolluted and organically enriched habitats in Florida at 0.25 kg/ha reduced populations ofCulex spp. by 93–100% and 99%, respectively. Sustained-release briquets (5% primary powder) applied at the rate of one half briquet/1.8 m² sod-lined potholes reduced larval populations ofCx. quinquefasciatus by 88–95% for up to 2 weeks in open sunlight. Sustained-release pellets (30% primary powder) applied to small woodland pools in Memphis, TN at the rate of four pellets/pool virtually eliminated larval populations ofCx. restuans Theobald for over 8 days. Variable persistance of larvicidal activity was noted for the other treatments depending on the formulation, target species and habitat.     

An immobilized fork as a termination of replication intermediate in Bacillus subtilis

The structure of a DNA intermediate associated with termination of chromosome replication in Bacillus subtilis and derived from a unique BamHI 24.8 X 10(3) base-pair (bp) region of the chromosome has been investigated. The intermediate has properties expected for a forked structure. Gel electrophoresis followed by Southern transfer and hybridization to cloned DNA has shown it to comprise single strands of 15.4 X 10(3) bp and 24.8 X 10(3) bp, in approximately equimolar amounts. After purification away from the bulk of chromosomal DNA, electron microscopy of the intermediate established that 15% of the DNA was present as branched molecules and a significant proportion (11 of 31) of these contained two arms of matching length. The average dimensions (best estimates) of this unique class of Y-shaped molecule were 9.5(+/- 0.3) X 10(3), 15.1(+/- 0.4) X 10(3) and 24.6 24.6(+/- 0.6) X 10(3) bp for the stem, arms and end-to-end length, respectively. These values are consistent with the single strand composition of the intermediate as found. Furthermore, hybridization of the single strands to DNA from known locations within the BamHI 24.8 X 10(3) bp region has established the orientation of the forked intermediate relative to the genetic map. The intermediate presumably reflects the immobilization of the clockwise replication fork within the 24.8 X 10(3) bp region, at a location approximately 15.4 X 10(3) bp from the right end.     

Structural Proteins of Adenovirus-Associated Viruses

The structural proteins of adenovirus-associated virus (AAV) types 1, 2, and 3 were analyzed by acrylamide gel electrophoresis. In each case, one major protein (C) and two minor proteins (A and B) were identified. Component C had an estimated molecular weight of 62,000 daltons, and the molecular weights of components A and B were found to be 87,000 and 73,000 daltons, respectively. Coelectrophoresis of adenovirus and AAV proteins revealed an overlap only between the adenovirus fiber-penton component and the AAV C polypeptide. Among AAV serotypes, homologous components were electrophoretically identical, except that the C component of AAV-2 was of slightly lower molecular weight than the C components of AAV-1 and AAV-3. The relative incorporation of (14)C-arginine and (14)C-mixed amino acids into the three polypeptides of AAV-2 was similar, indicating an absence of an arginine-rich component. In addition, AAV-2 was found to have a substantially lower arginine content than helper adenoviruses.