Two-dimensional polyacrylamide gel electrophoresis characterization of APz, a sperm protein involved in zona binding in the pig and evidence for its binding to specific zona glycoproteins

A boar sperm integral plasma membrane protein (APz) involved in the adhesion of uncapacitated and capacitated sperm to the porcine zona pellucida (ZP) has been characterized by two-dimensional polyacrylamide gel electrophoresis (PAGE) and tested for its ability to bind to various zona glycopeptides. APz shows microheterogeneity and focuses over a wide pH range, with predominant forms focusing above pH 7. The protein, when excised from nonreducing polyacrylamide gels, inhibited sperm-egg binding and bound heat-solubilized zonae preventing these zonae from blocking sperm binding to eggs. In an indirect assay, a polyclonal monovalent antibody, which blocks sperm-egg binding and which is absorbed by APz, was used to determine the ability of zona glycopeptides to prevent the sperm-egg blocking activity of the antibody from being absorbed by intact sperm. When whole heat-solubilized ZP was added to sperm at doses that block sperm-egg binding and the excess ZP was removed, the sperm-egg blocking activity of the antibody was not absorbed by these sperm, and antibody-containing supernatants blocked the binding of untreated sperm to eggs as effectively as antibody that was not mixed with fresh sperm. When alpha ZP3 was used in the same manner, sperm-egg blocking activity again was not absorbed by antibody-treated cells. Beta ZP3, however, failed to block sperm-egg binding and failed to absorb the sperm-egg blocking activity of the antibody. These findings support the argument that the action of APz is physiologically significant and involves specific binding sites on the ZP3 component of the ZP.     

Relationship between Net CO(2) Assimilation and Dry Weight Accumulation in Field-Grown Tobacco

To assess the variability of net photosynthetic CO₂ exchange per unit leaf area and to construct budgets for stands of field-grown tobacco (Nicotiana tabacum, Connecticut Broadleaf), a number of short-time measurements were made on all available leaf positions on two varieties using a hand-held transparent chamber for conducting gas exchange measurements on leaves. Measurements of net CO₂ exchange were carried out on 18 separate days during a 35-day period, beginning 22 days after the seedlings were transplanted to the field. Gas exchange assays on leaves were conducted under ambient conditions of temperature and light intensity at all times of day. Solar radiation was monitored throughout the period, and losses of respiratory CO₂ from stems, roots, and leaves (in the dark) were estimated. A simple model was proposed to relate daily total CO₂ input to irradiance and total leaf area. The total leaf area was assumed to be a function of day number. Dark respiratory losses accounted for 41% to 47% of total CO₂ assimilation. Analysis of variance indicated that the two varieties were not significantly different in whole plant rate of CO₂ fixation per unit of leaf area. CO₂ input was closely associated with leaf area within each variety. Throughout the experiment, the difference between the two varieties in total leaf area per plant was the largest single factor in determining net CO₂ inputs. The cumulative dry weight increase for each variety was similar to the prediction of net dry matter input obtained by gas exchange measurements, thus confirming the close relationship between total plant net CO₂ assimilation and dry weight yield.     

Response of traplining bumble bees to competition experiments: shifts in feeding location and efficiency

Summary In field experiments, we mapped the regular foraging routes (traplines) of marked bumble bees visiting Aralia hispida. When other bees were removed to create localized competitive vacuums, the marked bees shifted their feeding activity toward the removal areas. Bees foraging in these competitive vacuums probed more flowers per inflorescence than control bees. One bee's foraging was studied intensively before and after its local competitors were removed. Compared to four nestmates foraging elsewhere, the focal bee's trip times were shorter (p<0.005) and its food collection rate was marginally higher (P=0.064) during the removal, although all the bees foraged similarly before the removal. These observations indicate that traplining bumble bees opportunistically modify their use of space in response to the activities of other bees in a highly competitive environment.     

Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.     

Long distance translocation of sucrose, serine, leucine, lysine, and carbon dioxide assimilates: I. Soybean

To determine the selectivity of movement of amino acids from source leaves to sink tissues in soybeans (Glycine max [L.] Merr. `Wells'), ¹⁴C-labeled serine, leucine, or lysine was applied to an abraded spot on a fully expanded trifoliolate leaflet, and an immature sink leaf three nodes above was monitored with a GM tube for arrival of radioactivity. Comparisons were made with ¹⁴C-sucrose and ¹⁴CO₂ assimilates. Radioactivity was detected in the sink leaf for all compounds applied to the source leaflet. A heat girdle at the source leaf petiole essentially blocked movement of applied compounds, suggesting phloem transport. Transport velocities were similar (ranged from 0.75 to 1.06 cm/min), but mass transfer rates for sucrose were much higher than those for amino acids. Hence, the quantity of amino acids entering the phloem was much smaller than that of sucrose. Extraction of source, path, and sink tissues at the conclusion of the experiments revealed that 80 to 90% of the radioactivity remained in the source leaflet. Serine was partially metabolized in the transport path, whereas lysine and leucine were not. Although serine is found in greater quantities than leucine and lysine in the source leaf and path of soybeans, applied leucine and lysine were transported at comparable velocities and in only slightly lower quantities than was applied serine. Thus, no selective barrier against entry of these amino acids into the phloem exists.     

Charring does not affect wood infestation by subterranean termites

Fire is an important part of forest ecosystems, as is the insect fauna. Changes in wood brought about by fire may alter the ability of termites to use the wood, interrupting the decay cycle of woody debris. The ability of termites to find, infest, and feed upon wood after it had been charred was evaluated in the laboratory and field. Eastern subterranean termites, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae), fed on char from burned wood had significantly reduced numbers of protozoa compared to termites fed on pine shavings, but significantly more than starved termites. The ability of termites to find and infest wood was not affected by surface charring. In a laboratory choice test, there were no significant differences in the onset of feeding by termites between charred and non‐charred wood boards. Likewise in the field, no differences were observed in the time to initial attack by termites on charred and non‐charred wood boards or bolts. Because termites will likely survive fires of low to moderate intensity, in most cases, there should be no disruption of the termite contribution to forest nutrient and carbon cycles.     

Atmospheric CO2 concentration, N availability, and water status affect patterns of ergastic substance deposition in longleaf pine (Pinus palustris Mill.) foliage

 Leaf chemistry alterations due to increasing atmospheric CO₂ will reflect plant physiological changes and impact ecosystem function. Longleaf pine was grown for 20 months at two levels of atmospheric CO₂ (720 and 365 μmol mol^–1), two levels of soil N (4 g m^–2 year^–1 and 40 g m^–2 year^–1), and two soil moisture levels (– 0.5 and – 1.5 MPa) in open top chambers. After 20 months of exposure, needles were collected and ergastic substances including starch grains and polyphenols were assessed using light microscopy, and calcium oxalate crystals were assessed using light microscopy, scanning electron microscopy, and transmission electron microscopy. Polyphenol content was also determined using the Folin-Denis assay and condensed tannins were estimated by precipitation with protein. Evaluation of phenolic content histochemically was compared to results obtained using the Folin-Denis assay. Total leaf polyphenol and condensed tannin content were increased by main effects of elevated CO₂, low soil N and well-watered conditions. Elevated CO₂ and low soil N decreased crystal deposition within needle phloem. Elevated CO₂ had no effect on the percentage of cells within the mesophyll, endodermis, or transfusion tissue which contained visible starch inclusions. With respect to starch accumulation in response to N stress, mesophyll > endodermis > transfusion tissue. The opposite was true in the case of starch accumulation in response to main effects of water stress: mesophyll < endodermis < transfusion tissue. These results indicate that N and water conditions significantly affect deposition of leaf ergastic substances in longleaf pine, and that normal variability in leaf tissue quality resulting from gradients in soil resources will be magnified under conditions of elevated CO₂. Received: 5 November 1996 / Accepted: 7 March 1997     

Influence of acetaminophen consumption and exercise on Achilles tendon structural properties in male Wistar rats

Chronic consumption of acetaminophen (APAP) during exercise training leads to a reduction in tendon stiffness and modulus compared with a placebo. We explored whether this effect could be due to a reduction in tendon collagen content or cross-linking. Ten-week-old male Wistar rats (n = 50) were divided into placebo or APAP groups and into sedentary or treadmill-exercised groups. APAP (200 mg/kg) or saline was administered once daily by oral gavage. Rats in the exercise groups ran on a treadmill 5 days per week for 8 wk with progression to 60 min per day, 20 m/min, and 8° incline. After 8 wk, lyophilized Achilles tendon samples were assayed for the collagen-specific amino acid hydroxyproline and cross-linking [hydroxylyslpyridinoline (HP)] content by high-performance liquid chromatrography. Collagen content was not influenced by exercise or APAP (P > 0.05). Compared with placebo, tendon water content was 7% (P = 0.006, main effect) lower in animals consuming APAP (placebo: 54.79 ± 0.8%, APAP: 50.89 ± 1.2%). HP in the Achilles tendon was 36% greater (sedentary: 141 ± 15, exercise: 204 ± 26 mmol/mol collagen) in the exercise-trained rats independent of drug treatment (P = 0.020, main effect). Independent of exercise, HP content was 33% lower (P = 0.032, main effect) in the animals consuming APAP (placebo: 195 ± 21, APAP: 140 ± 19 mmol/mol collagen). Our data suggests that chronic consumption of APAP results in a reduction in collagen cross-linking and a loss of tissue water independent of chronic exercise. This reduction in cross-linking and water content could contribute to the decrease in tendon stiffness noted in humans chronically consuming APAP.