Odorant stimulation of secretory and neural processes in the salamander olfactory mucosa

Topical application of the odorants guaiacol (10(-3) mol/l, 1-30 min) and 2-isobutyl-3-methoxypyrazine (IBMP, 10(-5)-10(-3) mol/l, 15 min) caused time- and concentration-dependent reductions in the secretory granule content of acinar cells of the superficial Bowman's glands (sBG) and moderate to extensive vacuolation in acinar cells of sBG and deep olfactory glands (dG). Topical application of 9.8 mg/ml scopolamine 10 min before 10(-4) mol/l IBMP significantly reduced the amount of secretory granule depletion from sBG compared to that seen with IBMP alone and resulted in less extensive vacuolation in sBG and dG acinar cells. The i.p. injection of 42 mg/kg propranolol 10 min before topical application of 10(-4) mol/l IBMP had no effect on the action of IBMP. Guaiacol and IBMP also had time- and concentration-dependent effects on the secretory activity of sustentacular cells in the olfactory epithelium. The protrusion of secretory material into the mucociliary matrix that covers the epithelial surface and vacuolation within the secretory material resulted from odorant application. Scopolamine and propranolol had no effects on the action of IBMP on sustentacular cell secretory activity. When applied in the vapor phase, guaiacol elicited action potentials recorded from individual olfactory receptor neurons; the impulse frequency was concentration-dependent and showed tonic and phasic components when the duration of stimulation was varied. Low to moderate concentrations of IBMP delivered in the vapor phase evoked monophasic negative slow voltage transients recorded from the surface of the olfactory mucosa. The amplitudes of these transients increased with increasing stimulus concentrations. Higher concentrations or longer stimulus durations evoked longer-latency positive-voltage generating processes and negative afterpotentials. The properties of the electrophysiological responses to both odorants were characteristic of responses evoked by a wide variety of 'typical' odorants.     

Sugar-activated ion transport in canine lingual epithelium. Implications for sugar taste transduction

There is good evidence indicating that ion-transport pathways in the apical regions of lingual epithelial cells, including taste bud cells, may play a role in salt taste reception. In this article, we present evidence that, in the case of the dog, there also exists a sugar-activated ion-transport pathway that is linked to sugar taste transduction. Evidence was drawn from two parallel lines of experiments: (a) ion-transport studies on the isolated canine lingual epithelium, and (b) recordings from the canine chorda tympani. The results in vitro showed that both mono- and disaccharides in the mucosal bath stimulate a dose-dependent increase in the short-circuit current over the concentration range coincident with mammalian sugar taste responses. Transepithelial current evoked by glucose, fructose, or sucrose in either 30 mM NaCl or in Krebs-Henseleit buffer (K-H) was partially blocked by amiloride. Among current carriers activated by saccharides, the current response was greater with Na than with K. Ion flux measurements in K-H during stimulation with 3-O-methylglucose showed that the sugar-evoked current was due to an increase in the Na influx. Ouabain or amiloride reduced the sugar-evoked Na influx without effect on sugar transport as measured with tritiated 3-O-methylglucose. Amiloride inhibited the canine chorda tympani response to 0.5 M NaCl by 70-80% and the response to 0.5 M KCl by approximately 40%. This agreed with the percent inhibition by amiloride of the short-circuit current supported in vitro by NaCl and KCl. Amiloride also partially inhibited the chorda tympani responses to sucrose and to fructose. The results indicate that in the dog: (a) the ion transporter subserving Na taste also subserves part of the response to K, and (b) a sugar-activated, Na-preferring ion-transport system is one mechanism mediating sugar taste transduction. Results in the literature indicate a similar sweet taste mechanism for humans.     

The biochemical identification of fibronectin in the sea urchin embryo

We report the biochemical identification of fibronectin in the basal lamina of the sea urchin embryo. A. punctulata gastrula stage embryos were solubilized in Triton X-100 and the insoluble basal laminae extracted by incubation in buffer containing 8M urea, 2% 2-mercaptoethanol and 2% SDS. Extracted proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose filters and probed with monospecific antibodies directed against human plasma fibronectin (pFN). Incubation in 125I-labelled secondary antibody revealed a single band which co-migrates with human pFN at an apparent molecular weight of 220,000. This is the first direct biochemical demonstration of a fibronectin-like molecule in the sea urchin embryo which cross reacts with antibodies to vertebrate fibronectin.     

The identity of the current carriers in canine lingual epithelium in vitro

Ion transport across the lingual epithelium has been implicated as an early event in gustatory transduction. The fluxes of isotopically labelled Na+ and Cl- were measured across isolated canine dorsal lingual epithelium under short-circuit conditions. The epithelium actively absorbs Na+ and to a lesser extent actively secretes Cl-. Under symmetrical conditions with Krebs-Henseleit buffer on both sides, (1) Na+ absorption accounts for 46% of the short-circuit current (Isc); (2) there are two transcellular Na+ pathways, one amiloride-sensitive and one amiloride-insensitive; (3) ouabain, added to the serosal solution, inhibits both Isc and active Na+ absorption. When hyperosmotic (0.25 M) NaCl is placed in the mucosal bath, both Isc and Na+ absorption increase; net Na+ absorption is at least as much as Isc. Ion substitution studies indicate that the tissue may transport a variety of larger ions, though not as effectively as Na+ and Cl-. Thus we have shown that the lingual epithelium, like other epithelia of the gastrointestinal tract, actively transports ions. However, it is unusual both in its response to hyperosmotic solutions and in the variety of ions that support a transepithelial short-circuit current. Since sodium ion transport under hyperosmotic conditions has been shown to correlate well with the gustatory neural response, the variety of ions transported may likewise indicate a wider role for transport in taste transduction.     

Cellular Concentrations and Uniformity of Cell-Type Accumulation of Two Lea Proteins in Cotton Embryos

The levels and cell-type distribution of late embryogenesis abundant (Lea) proteins D-7 and D-113 have been determined in mature cotton embryos by immunochemical methods. The two proteins were expressed in and purified from Escherichia coli and utilized for antibody production in rabbits. The antiserum to each protein was found to interact with all members of each protein family in cotton extracts by protein gel blotting. Using these antibodies in quantitative "rocket" immunoelectrophoreses, D-7 proteins were found to accumulate to ~8 x 1015 molecules per embryo, which is equivalent to ~109 molecules per "average cell." D-113 proteins accumulate to ~1016 molecules per embryo, which equates to ~1.3 x 109 molecules per average cell. These values calculate to concentrations of about 226 and 283 [mu]M, respectively, in the cell aqueous phase immediately prior to seed desiccation. In immunocytochemical studies using the fluorophor rhodamine linked to the secondary antibody, both proteins appeared to be evenly present in the cytosol of all cell types present in the embryo, including both cotyledon and axis epidermal cells. Thus, their function does not appear related to unique functions of specific cell or tissue types. The very high molar concentrations of the two proteins, coupled with their unusual predicted structure and their cytosol location, would seem to reduce the number of their conceivable functions.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

Hemoglobin synthesis studies of a family with α-thalassemia trait and sickle cell trait

The ratio of total globin alpha to beta chain synthesis was determined in reticulocytes isolated from the blood of the members of a black family, some of whom had sickle cell trait with low blood HbS concentrations (25-30%). The results support the hypothesis that sickle cell trait individuals with low HbS concentrations also carry a gene for alpha-thalassemia.     

Restoration and Science: A Practitioner/Scientist's View from Rare Habitat Restoration at a Southern California Preserve

Researchers reexamining the relationship between restoration science and practice report a continuing scientist‐practitioner gap. As a land manager with scientific training, I offer my perspective of the chasm and describe a restoration practice infused with as much science as the realities of limited budget and time allow. The coastal sage scrub (CSS) restoration project at Starr Ranch, a 1,585 ha Audubon preserve in southern California, combines non‐chemical invasive species control, restoration, and applied research. Our practices evolve from modified scientific approaches and the scientific literature. Results from experiments with non‐optimum replication (on effects of seed rates, soil tamping, and timing of planting) nonetheless had value for management decisions. A critical practice came from academic research that encouraged cost‐effective passive restoration. Our passive restoration monitoring data showed 28–100% total native cover after 3–5 years. Another published study found that restoration success in semiarid regions is dependent on rainfall, a finding vital for understanding active restoration monitoring results that showed a range of 0–88% total native cover at the end of the first season. Work progresses through a combination of applied research, a watchful eye on the scientific literature, and “ecological intuition” informed by the scientific literature and our own findings. I suggest that it is less critical for academic scientists to address the basic questions on technique that are helpful to land managers but rather advocate practitioner training in methods to test alternative strategies and long‐term monitoring.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.