Seasonal activity and energetics of two species of varanid lizards in tropical Australia

The field metabolic rates (FMR) and rates of water flux were measured in two species of varanid lizards over five periods of the year in tropical Australia. The energetics of these species were further investigated by directly measuring activity (locomotion) and body temperatures of free-ranging animals by radiotelemetry, and by measuring standard metabolic rate (over a range of body temperatures) and activity metabolism in the laboratory. Seasonal differences in the activity and energetics were found in these goannas despite similar, high daytime temperatures throughout the year in tropical Australia. Periods of inactivity were associated with the dry times of the year, but the onset of this period of inactivity differed with respect to habitat even within the same species. Varanus gouldii, which inhabit woodlands only, were inactive during the dry and late dry seasons. V. panoptes that live in the woodland had a similar seasonal pattern of activity, but V. panoptes living near the floodplain of the South Alligator River had their highest levels of activity during the dry season when they walked long distances to forage at the receding edge of the floodplain. However, during the late dry season, after the floodplain had dried completely, they too became inactive. For V. gouldii, the rates of energy expenditure were 196 kJ kg^–1 day^–1 for active animals and 66 kJ kg^–1 day^–1 for inactive animals. The rates of water influx for these groups were respectively 50.7 and 19.5 ml kg^–1 day^–1. For V. panoptes, the rates of energy expenditure were 143 kJ kg^–1 day^–1 for active animals and 56 kJ kg^–1 day^–1 for inactive animals. The rates of water influx for these two groups were respectively 41.4 and 21.0 ml kg^–1 day^–1. We divided the daily energy expenditure into the proportion of energy that lizards used when in burrows, out of burrows but inactive, and in locomotion for the two species during the different seasons. The time spent in locomotion by V. panoptes during the dry season is extremely high for a reptile (mean of 3.5 h/day spent walking), and these results provide an ecological correlate to the high aerobic capacity found in laboratory measurements of some species of varanids.     

Partitioning of water resources among plants of a lowland tropical forest

Source water used by plants of several species in a semi-evergreen lowland tropical forest on Barro Colorado Island, Panama, was assessed by comparing the relative abundance of deuterium, D, versus hydrogen, H (stable hydrogen isotope composition, D) in xylem sap and in soil water at different depths, during the dry season of 1992. Ecological correlates of source water were examined by comparing xylem water D values with leaf phenology, leaf water status determined with a pressure chamber, and rates of water use determined as mass flow of sap using the stem heat balance method. Soil water D values decreased sharply to 30 cm, then remained relatively constant with increasing depth. Average D values were-13, for 0–30 cm depth and-36.7 for 30–100 cm depth. Soil water D values were negatively associated with soil water content and soil water potential. Concurrent analyses of xylem water revealed a high degree of partitioning of water resources among species of this tropical forest. Xylem water D of deciduous trees (average=-25.3±1.4) was higher than that of evergreen trees (average=-36.3±3.5), indicating that evergreen species had access to the more abundant soil water at greater depth than deciduous species. In evergreen shade-tolerant and high-light requiring shrubs and small trees, D of xylem water was negatively correlated with transpiration rate and leaf water potential indicating that species using deeper, more abundant water resources had both higher rates of water use and more favorable leaf water status.     

Purification and characterization of a 22-kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica . The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22-kDa protein was isolated from thylakoid membranes of spores and the amino-terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS-PAGE and by the disappearance of the band during spore germination. Protease activity against the 22-kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.     

Transport of bacterial inoculants through intact cores of two different soils as affected by water percolation and the presence of wheat plants

Abstract Water flow-innduced transport of Burkholderia cepacia strain P2 and Pseudomonas fluorescens strain R2f cells through intact cores of loamy sand and silt loam field soils was measured for two percolation regimes, 0.9 and 4.4 mm h⁻¹, applied daily during 1 hour. For each strain, transport was generally similar between the two water regimes. Translocation of B. cepacia , with 4.4 mm h⁻¹, did occur initially in both soils. In the loamy sand soil, no change in the bacterial distribution occurred during the experiment (51 days). In the silt loam, B. cepacia cell numbers in the lower soil layers were significantly reduced, to levels at or below the limit of detection. Transport of P. fluorescens in both soils also occurred initially and was comparable to that of B. cepacia . Later in the experiment, P. fluorescens was not detectable in the lower soil layers of the loamy sand cores, due to a large decrease in surviving cell numbers. In the silt loam, the inoculant cell distribution did not change with time. Pre-incubation of the inoculated cores before starting percolation reduced B. cepacia inoculant transport in the loamy sand soil measured after 5 days, but not that determined after 54 days. Delayed percolation in the silt loam soil affected bacterial transport only after 54 days. The presence of growing wheat plants overall enhanced bacterial translocation as compared to that in unplanted soil cores, but only with percolating water. Percolation water from silt loam cores appeared the day after the onset of percolation and often contained inoculant bacteria. With loamy sand, percolation water appeared only 5 days after the start of percolation, and no inoculant bacteria were found. The results presented aid in predicting the fate of genetically manipulated bacteria in a field experiment.     

Purification and structural characterization of insulin from a caecilian, Typhlonectes natans (Amphibia: Gymnophiona)

Despite the important position of amphibia in phylogeny, efforts at the structural characterization of amphibian neurohormonal peptides have largely been confined to the Anurans (frogs and toads). Insulin was purified from an extract of the pancreas of the caecilian, Typhlonectes natans. The primary structure of the peptide was established as:

A-chain: Gly-Ile-Val-Glu-Lys⁵-Cys-Cys-Leu-Ser-Thr¹⁰-Cys-Ser-Leu-Tyr-Glu¹⁵-Leu-Glu-Ser-Tyr-Cys²⁰-Asn

B-chain: Ile-Ala-Asn-Gln-His⁵-Leu-Cys-Gly-Ser-His¹⁰-Leu-Val-Glu-Ala-Leu¹⁵-Tyr-Leu-Val-Cys-Ala²⁰-Asp-Arg-Gly-Phe- Phe²⁵-Tyr-Thr-Pro-Lys-Ser³⁰

This amino acid sequence contains several unusual substitutions (Gln → Lys at A5, His → Leu at A8, Gln → Glu at A15, and Gly → Ala at B20) that are not present in other amphibian insulins. The structure of insulin appears to be less well conserved among the different orders of amphibia, compared with reptiles and birds.     

The paracellular channel for water secretion in the upper segment of the Malpighian Tubule of Rhodnius prolixus

Lumen to bath J ₁₂/C ₁ and bath to lumen J ₂₁/ C ₂ fluxes per unit concentration of 19 probes with diameters (d ^m) ranging from 3.0–30.0 Å (water, urea, erythritol, mannitol, sucrose, raffinose and 13 dextrans with d ^m 9.1–30.0 Å) were measured during volume secretion (J _v ) in the upper segment of the Malpighian Tubule of Rhodnius by perfusing lumen and bath with ¹⁴C or ³H-labeled probes. J _net=(J ₁₂/C ₁ – J ₂₁/C ₂) was studied as a function of J _v · J _v was varied by using different concentrations of 5-hydroxy tryptamine. J _net for ³H-water was not different from J _v We found: (i) A strong correlation between J _net and J _v for 8 probes d ^m =3.0–11.8 Å (group a probes), indicating that the convective component of J _net is more important than its diffusive component and than unstirred layers effects which are negligible. Therefore group a probes are solvent dragged as they cross the epithelium, (ii) There is no correlation between J _net and J _v for 11 probes with d ^m=11.8–30 Å (group b). Therefore these probes must cross the epithelium by diffusion and not by solvent drag, (iii) In a plot of J _net/J _v vs. d ^m group a probes show a steep linear relation with a slope = –0.111, while for group b probes the slope is –0.002. Thus there is a break between groups a and b in this plot. We tried to fit the data with models for restricted diffusion and convention through cylindrical or parallel slit pathways. We conclude that (i) group a probes are dragged by water through an 11.0 Å-wide slit, (ii) Most of J _v must follow an extracellular noncytosolic pathway, (iii) Group b probes must diffuse through a 42 Å-wide slit, (iv) A cylindrical pathway does not fit the data.E.G. is a Visiting Scientist at IVIC. It is a pleasure to thank Drs. A.E. Hill and Bruria Shachar-Hill for their suggestion of the use of dextrans, their instruction and help with the dextran separation technique, and their extensive discussions. Dr. R. Apitz, Mr H. Rojas and Mrs. Fulvia Bartoli were most helpful with suggestions during the course of the experimental work. Mr. Jose Mora was fundamental help with the equipment. Mrs. Lelis Ochoa and Mr. Luis F. Alvarez helped with some of the drawings. This work was partially supported by CONICIT, Fundación Polar and CDCH of UCV. It is a pleasure to thank Dr. H. Passow and Dr. K.J. Ullrich at the Max Planck Institut für Biophysik (Frankfurt/Main) where this work was initiated.     

Chloride-activated water permeability in the frog corneal epithelium

We have previously reported that the isolated frog corneal epithelium (a Cl^–-secreting epithelium) has a large diffusional water permeability (P_dw 1.8×10^–4 cm/s). We now report that the presence of Cl^– in the apical-side bathing solution increases the diffusional water flux, J_dw (in both directions) by 63% from 11.3 to 18.4 l min^–1 · cm^–2 with 60 mm [Cl] exerting the maximum effect. The presence of Cl^– in the basolateral-side bathing solution had no effect on the water flux. In Cl^–-free solutions amphotericin B increased J_dw by 29% but only by 3% in Cl^–-rich apical-side bathing solution, suggesting that in Cl^–-rich apical side bathing solution, the apical barrier is no longer rate limiting. Apical Br^– (75 mm) also increased J_dw by 68%. The effect of Cl^– on J_dw was observed within 1 min after its addition to the apicalside bathing solution. HgCl₂ (0.5 mm) reduced the Cl^–-increased P_dw by 31%. The osmotic permeability (P_f) was also measured under an osmotic gradient yielding values of 0.34 and 2.88 (x 10^–3 cm/s) in Cl^–-free and Cl^–-rich apical-side bathing solutions respectively. It seems that apical Cl^–, or Cl^– secretion into the apical bath could activate normally present but inactive water channels. In the absence of Cl^–, water permeability of the apical membrane seems to be limited to the permeability of the lipid bilayer.This work was supported by National Eye Institute grants EY-00160 and EY-01867.     

Expression of a novel sodium-hydrogen exchanger in the gastrointestinal tract and kidney

Aquaporin CHIP, a 28 kDa channel forming protein, has been proposed to function as water channel in both erythrocyte and kidney proximal tubule. Recently, we have reported that in frog urinary bladder, a model of the kidney collecting tubule, polyclonal antibodies against human erythrocyte CHIP recognize and immunoprecipitate a 30 kDa protein from the epithelial cell homogenate. In the present work confocal fluorescence microscopy was used to determine the cellular and subcellular localization of CHIP28-like proteins in the urinary epithelium. A clear labeling of the apical border was found after Triton X-100 permeabilization. The labeling was distributed throughout the apical domain and not restricted to specific domains of the membrane. The staining was also present in the deeper confocal sections where the fluorescence seems to be localized at the cellular contour. No difference in the labeling patterns was observed between resting and ADH-treated bladder. Specificity of the staining was confirmed by the absence of the labeling pattern when antiserum was preadsorbed on CHIP28 protein immobilized on Immobilon P stripes. Our results suggest that CHIP-like proteins are not proteins inserted in the apical membrane during the antidiuretic response. Moreover, we do not know whether the labeling was due to the presence of CHIP28 itself or an as-yet-unidentified protein sharing immunological analogies with aquaporin CHIP.     

Ion binding constants for gramicidin a obtained from water permeability measurements

Gramicidin A pores are permeable to water and small monovalent cations. For K, Rb, and Cs there is good evidence from conductances and permeability ratios that a second ion can enter a pore already occupied by another, but for Na this evidence is inconclusive and comparison of tracer fluxes and single channel conductances suggests that second ion entries are prohibited. Partly as a result of the complications of second ion entry there have been widely differing estimates for the dissociation constants for the first ion in the channel. Dani and Levitt (1981, Biophys. J. 35: 485–499) introduced a method for calculating ion binding constants from simultaneous measurements of water fluxes and membrane conductance. They found no evidence for second ion binding and calculated dissociation constants of 115 mm for Li, 69 mm for K, and 2 mm for Tl. It is shown here that the two-ion, four-state model predicts a dependence of water permeability on ion concentration that is difficult to distinguish from the predictions of block by a single ion. Using a modified technique that allows measurement of higher conductances, the first ion dissociation constants have been determined as 80 mm for Na, 40 mm for Rb and 15 mm for Cs. These values and those of Dani and Levitt fall in a smooth sequence. The dissociation constant for Cs is consistent with single channel conductances and flux ratios. There is a discrepancy between this constant for Na and the value, 370 mm, calculated from the single channel conductances and the assumption that a second ion cannot enter or affect an occupied pore. The dissociation constant for Rb is intermediate between those for K and Cs whereas tracer flux measurements (Schagina, Grinfeldt & Lev, 1983. J. Membrane Biol. 73: 203–216) have suggested that Rb interacts much more strongly with the channel than Cs.We should like to thank the National Grid plc, for the grant which supported K.-W.W., the Wellcome Trust for a visiting Fellowship for S.T. in Cambridge, and the Cambridge Society of Bombay which supported S.B.H. in Bombay.