The dissociation of the fluid and particle phase in the forestomach as a physiological characteristic of large grazing ruminants: an evaluation of available, comparable ruminant passage data

Whether differences in digestive physiology exist between different ruminant feeding types has been an ongoing debate. In this regard, potential differences in ingesta retention have been understood to be of particular importance. We analyzed a data pool in which only mean retention time (MRT) data for the ruminoreticulum (RR) were collated that were obtained using comparable techniques with either chromium or cobalt EDTA as a fluid marker and/or with chromium-mordanted fiber of less than 2 mm in size as a particle marker. Data were compared using one averaged value per species. In general, the paucity of species in such a collection is striking and does not allow—in contrast to earlier statements—any final conclusions regarding the influence of body weight (BW) or feeding type on ruminant MRTs. In particular, there was no significant correlation between MRT_particlesRR or MRT_fluidRR and BW, neither in the interspecific nor in the intraspecific comparisons, and no difference between the feeding types. The trend that indicates longer MRT_particlesRR in grazers is based on too few species to be conclusive. Small browsers seemed to have shorter MRT_fluidRR than similar-sized grazers. In contrast, there was a trend for large grazers to have shorter MRT_fluidRR than large browsers. In direct pair-wise comparisons between cattle and the browsers giraffe, moose, and okapi, the latter difference was significant. Cattle also had the highest relative RR fluid outflow rates among the species investigated. This is in accord with the observation that grazers have larger omasa, a major function of which is water-reabsorption distal to the RR. Grazers seem to have longer MRT_particlesRR per unit MRT_fluidRR, and cattle are particular outliers in this respect. It is hypothesized that potentially shorter MRT_fluidRR in large grazers and higher relative outflow rates are linked to a higher saliva production and a lesser viscosity of both saliva and RR fluids. A constant supply of a fluid phase of low viscosity is proposed to be the prerogative for the physical mechanisms of flotation and sedimentation that result in the stratification of RR contents and its selective particle retention typical for large grazing species.     

Retention of solute and particle markers in the digestive tract of captive Somali wild asses (<Emphasis Type="Italic">Equus africanus somaliensis</Emphasis>)

In contrast to the domestic horse, whose digestive physiology has been thoroughly investigated, knowledge on the digestive physiology of wild equids is scarce. Comparisons between the domestic horse and the domestic donkey suggest that wild asses might achieve higher digestibilities. This could derive from longer retention times or a greater difference in the mean retention time (MRT) of particles vs. fluid (the selectivity factor (SF)). Here, we measured MRT of a solute (fluid; MRT_solute) and a particle (<2 mm; MRT_particle) marker in five captive male Somali wild asses (Equus africanus somaliensis) fed a diet of 95% grass hay. At a mean dry matter intake of 94 ± 3 g kg^?0.75 day^?1, MRT_solute was 33.3 ± 5.4 h and MRT_particle 39.6 ± 3.9 h, resulting in a SF of 1.21 ± 0.14. For their food intake, Somali wild asses appeared to have slightly higher MRT_particle than expected based on domestic equid data, in contrast to Grevy zebras (Equus grevyi), potentially indicating higher capacities of the digestive tract. However, considering data on domestic horses, donkeys, and zebra, there was no evident difference in the SF of wild equids compared to domestic ones. Together with an absence of reported anatomical differences in the digestive tract of wild and domestic equids, the data suggest a general similarity in the digestive physiology of equid species that contrasts with the diversity in the digestive physiology of ruminants, and that might be one contributing factor to a lack of sympatric, niche-differentiated equid species.     

Seasonal faecal excretion,gut fill,liquid and particle marker retention in mouflon<Emphasis Type="Italic">Ovis ammon musimon</Emphasis>, and a comparison with roe deer<Emphasis Type="Italic">Capreolus capreolus</Emphasis>

Five mouflon [average body mass (BM) 33 kg] and two roe deer (average BM 20 kg) with rumen cannulas were kept in large enclosures under semi-natural conditions and were used for seasonal studies on gastrointestinal tract (GIT) indigestible fill and digesta passage kinetics. As the mouflon were not fully mature, both species had similar digesta volumes in the reticulorumen (RR; mouflon 5.5 ± 1.8% of BM; roe deer 5.4 ± 1.5% of BM); however, the mouflon had lower RR liquid flow rates (15.1 ± 4.3 ml h⁻¹ kg^−0.75) than the roe deer (19.2 ± 0.2 ml h⁻¹ kg^−0.75), and particle retention in the RR accounted for 68 ± 3% of total GIT retention in the mouflon versus 55 ± 6% in the roe deer. Annual average total GIT retention times for liquids and particles were longer in the mouflon (23.4 ± 0.9 h and 37.9 ± 4.0 h) than in the roe deer (18.4 ± 1.7 h and 22.4 ± 1.9 h). Similarly, annual average RR retention times for liquids and particles were longer in the mouflon (11.9 ± 0.9 h and 25.8 ± 3.3 h) than in the roe deer (8.1 ± 1.7 h and 12.5 ± 2.3 h). The factor of selective particle retention in the RR (retention of particles/retention of liquid) was 2.10 ± 0.09 in the mouflon versus 1.54 ± 0.01 in the roe deer. These observations are in accord with differences in digesta passage characteristics postulated between browsing and grazing ruminants. Total GIT indigestible fill was lower in the mouflon than in the roe deer (10.7 ± 2.1 g kg⁻¹ and 13.3 ± 1.0 g kg⁻¹).     

Fluid and particle retention in the digestive tract of the addax antelope (Addax nasomaculatus)--adaptations of a grazing desert ruminant.

Retention time of food in the digestive tract is a major aspect describing the digestive physiology of herbivores. Differences in feed retention times have been described for different ruminant feeding types. In this study, a dominantly grazing desert ruminant, the addax (Addax nasomaculatus), was investigated in this respect. Eight animals with a body weight (BW) of 87+/-5.3 kg on an ad libitum grass hay (Chloris gayana) diet were available. Co-EDTA and Cr-mordanted fibers (<2 mm) were used as pulse-dose markers. Mean retention time (MRT) in the digestive tract was calculated from faecal marker excretion. Average daily intake of the addax was found to be 1.7 kg dry matter (DM) or 60+/-8.3 g DM/kg BW(0.75). The MRT of fluid and particles in the reticulo-rumen (MRT(fluid)RR and MRT(particle)RR) were quantified to be 20+/-5.8 and 42+/-7.0 h respectively. When compared to literature data, MRT(fluid)RR was significantly longer than in cattle species, and MRT(particle)RR was significantly longer than in 11 taxa of all feeding types. The ratio of MRT(particle)RR/MRT(fluid)RR (2.3+/-0.5) was found to be within the range described for grazing ruminants. The long retention times found in the addax can be interpreted as an adaptation to a diet including a high proportion of slow fermenting grasses, while the long retention time of the fluid phase can be interpreted as a consequence of water saving mechanisms of the desert-adapted addax with a potentially low water turnover and capacious water storing rumen.     

Fluid and particle retention in the digestive tract of the addax antelope (Addax nasomaculatus)--adaptations of a grazing desert ruminant

Retention time of food in the digestive tract is a major aspect describing the digestive physiology of herbivores. Differences in feed retention times have been described for different ruminant feeding types. In this study, a dominantly grazing desert ruminant, the addax (Addax nasomaculatus), was investigated in this respect. Eight animals with a body weight (BW) of 87+/-5.3 kg on an ad libitum grass hay (Chloris gayana) diet were available. Co-EDTA and Cr-mordanted fibers (<2 mm) were used as pulse-dose markers. Mean retention time (MRT) in the digestive tract was calculated from faecal marker excretion. Average daily intake of the addax was found to be 1.7 kg dry matter (DM) or 60+/-8.3 g DM/kg BW(0.75). The MRT of fluid and particles in the reticulo-rumen (MRT(fluid)RR and MRT(particle)RR) were quantified to be 20+/-5.8 and 42+/-7.0 h respectively. When compared to literature data, MRT(fluid)RR was significantly longer than in cattle species, and MRT(particle)RR was significantly longer than in 11 taxa of all feeding types. The ratio of MRT(particle)RR/MRT(fluid)RR (2.3+/-0.5) was found to be within the range described for grazing ruminants. The long retention times found in the addax can be interpreted as an adaptation to a diet including a high proportion of slow fermenting grasses, while the long retention time of the fluid phase can be interpreted as a consequence of water saving mechanisms of the desert-adapted addax with a potentially low water turnover and capacious water storing rumen.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.     

Quantification of nicotine, chlorpyrifos and their metabolites in rat plasma and urine using high-performance liquid chromatography

A method was developed for the separation and quantification of the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate), its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate) and TCP (3,5,6-trichloro-2-pyridinol), the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method is based on using solid-phase extraction and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 210 and 280 nm. The compounds were separated using a gradient of 1–85% acetonitrile in water (pH 3.20) at a flow-rate ranging between 1 and 1.7 ml/min over a period of 15 min. The retention times ranged from 5.4 to 13.2 min. The limits of detection ranged between 20 and 150 ng/ml, while the limits of quantitation were between 150 and 200 ng/ml. Average percentage recovery of five spiked plasma samples was 80.2±7.9, 74.9±8.5, 81.7±6.9, 73.1±7.8, 74.3±8.3, 80.8±6.6, 81.6±7.3 and 81.4±6.5, and from urine 79.4±6.9, 77.8±8.4, 83.3±6.6, 72.8±9.0, 76.3±7.7, 83.4±7.9, 81.6±7.9 and 81.8±6.8 for chlorpyrifos, chlorpyrifos-oxon, TCP, pyridostigmine bromide, N-methyl-3-hydroxypyridinium bromide, DEET, m-toluamide and m-toluic acid, respectively. The relationship between peak areas and concentration was linear over a range between 200 and 2000 ng/ml.     

Digesta passage and functional anatomy of the digestive tract in the desert tortoise (Xerobates agassizii)

The herbivorous tortoise Xerobates agassizii contents with large fluctuations in the quality and abundance of desert pastures. Responses to grass (Schismus barbatus), herbage (Sphaeralcea ambigua) and pelleted diets were studied in captive animals. Digestive anatomy was investigated in wild tortoises. Cornified esophageal epithelia and numerous mucus glands along the digestive tract indicated a resistance to abrasive diets. Gastric contents were acidic whereas hindgut digesta were near neutral pH. The colon was the primary site of fermentation with short-chain fatty acids mainly comprised of acetate (69–84%), propionate (10–15%) and n-butyrate (1–12%). Fibre digestion was extensive and equivalent to 22–64% of digestible energy intakes. Large particles of grass (25 mm Crmordants) were excreted as a pulse but retained longer than either fluids (Co-EDTA) or fine particles (2 mm; Yb). Patterns of marker excretion suggested irregular mixing of only the fluid and fine particulate digesta in the stomach and the colon. Mean retention times of Crmordants were 14.2–14.8 days on the grass and highfibre pellets. Intakes of grass were low and accompanied by smaller estimates of digesta fill than for the high-fibre pellets. Digestive capacity was large and estimated at 11–21% of body mass on these diets. The capacious but simple digestive anatomy of the tortoise may provide the greatest flexibility in utilizing a variety of forages in its unreliable habitat.Abbreviations bm body mass - DM drymatter - EDTA ethylene-diamine tetra-acetic acid - MRT mean retention time - NDF neutral detergent fibre - SCFA short-chain fatty acid(s) - T _max time to maximum marker concentration     

Liver Freezing Response of the Freeze-Tolerant Wood Frog, Rana sylvatica, in the Presence and Absence of Glucose. II. Mathematical Modeling

The “two-step” low-temperature microscopy (equilibrium and dynamic) freezing methods and a differential scanning calorimetry (DSC) technique were used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing, in Part I of this study. In this study, the experimentally determined dynamic water transport data are curve fit to a model of water transport using a standard Krogh cylinder geometry (Model 1) to predict the biophysical parameters of water transport: L_pg = 1.76 μm/min-atm and E_Lp = 75.5 kcal/mol for control liver cells and L_pg[cpa] = 1.18 μm/min-atm and E_Lp[cpa] = 69.0 kcal/mol for liver cells equilibrated with 0.4 M glucose. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5°C/min but do so when cooled at 2°C/min. Cells also retained twice as much intracellular fluid in the presence of 0.4 M glucose than in control tissue when cooled at 5°C/min. The ability of R. sylvatica liver cells to retain water during fast cooling (≥5°C/min) appears to be primarily due to its liver tissue architecture and not to a dramatically lower permeability to water, in comparison to mammalian (rat) liver cells which do dehydrate completely when cooled at 5°C/min. A modified Krogh model (Model 2) was constructed to account for the cell–cell contact in frog liver architecture. Using the same biophysical permeability parameters obtained with Model 1, the modified Krogh model (Model 2) is used in this study to qualitatively explain the experimentally measured water retention in some cells during freezing on the basis of different volumetric responses by cells directly adjacent to vascular space versus cells at least one cell removed from the vascular space. However, at much slower cooling rates (1–2°C/h) experienced by the frog in nature, the deciding factor in water retention is the presence of glucose and the maintenance of a sufficiently high subzero temperature (≥−8°C).     

o-Phthalaldehyde–N-acetylcysteine polyamine derivatives: formation and stability in solution and in C18 supports

A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10⁻² min⁻¹ in solution versus 7.7±1.1×10⁻⁴ min⁻¹ on the (C₁₈) solid support. The results obtained showed that forming the derivative on the packing support (C₁₈) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples.     

Application of "in vivo cryotechnique" to detect erythrocyte oxygen saturation in frozen mouse tissues with confocal Raman cryomicroscopy

To measure oxygen saturation (SO₂) of flowing erythrocytes in blood vessels of living animals, our “in vivo cryotechnique” (IVCT) was combined with confocal Raman microscopy at low temperature (−150 °C), referred to as cryomicroscopy. We evaluated two resonance Raman (RR) shifts around 1355 and 1378 cm⁻¹, reflecting de-oxygenated and oxygenated hemoglobin molecular structures, respectively. Judging from the calibration analyses of quickly frozen human whole blood for the control experiment in vitro, the two RR shifts were well retained at the low temperature, and their calculated ratios mostly reflected the relative SO₂ measured with a blood-gas analyzer. In blood vessels of living mouse organs prepared with the IVCT, their RR spectral peaks were also detected at the same RR shifts obtained in human blood. In the blood vessels of living mouse small intestines, some arterioles and venules were clearly distinguishable by monitoring different peak patterns of their RR shifts. The different ratios of the RR shift-areas were calculated even in the arterial vessels. In blood vessels of mouse livers, the Raman spectra showed a lower peak shift of 1378 cm⁻¹ compared to that of 1355 cm⁻¹, indicating an SO₂ decrease in hepatic blood circulation. Thus, the new cryopreparation technique will enable us to directly analyze the in vivo SO₂ in various tissues of a whole animal body prepared with the IVCT, reflecting their living states.     

A preliminary study on the seasonal body temperature rhythms of the captive mountain hare (Lepus timidus)

The mountain hare (Lepus timidus) is a year-round active herbivore adapted to survive the boreal winter. Captive mountain hares (N = 4) were implanted with intraabdominal thermosensitive loggers to record their core body temperature (T_b) for a year and during food deprivation (8–48 h) in summer and winter. The average T_b was 38.7 ± 0.01 °C in summer and 38.3 ± 0.01 °C in winter. The yearly T_b correlated positively with the ambient temperature. The 24-h T_b was the highest from late scotophase to early photophase in summer and winter and the lowest during middle-late photophase in summer or during early-middle scotophase in winter. The range of the 24-h oscillations in T_b increased in three animals in winter. Food deprivation did not induce hypothermia in summer or winter. These preliminary data suggest that the mountain hare can spare a modest amount of energy with the wintertime reduction in T_b.     

Colorimetric determination of hydroxyurea in human serum using high-performance liquid chromatography

A high-performance liquid chromatography assay for hydroxyurea in human serum was developed based on a commercial colorimetric assay kit for urea (Sigma Diagnostics). Serum (0.5 ml), spiked with methylurea as an internal standard, was treated with 70% perchloric acid. Supernatant (0.2 ml) was combined with 0.7 ml of BUN acid reagent and 0.6 ml of BUN color reagent. The resulting colored reactant (100 μl) was analyzed on a 300×3.9 mm Bondclone 10 C₁₈ column coupled with a UV–Vis detector, at 449 nm. The mobile phase was 13% acetonitrile in water. Retention times of colored derivatives of hydroxyurea and methylurea were 6.5 and 12.2 min, respectively. The log–log calibration curve was linear from 0.0065 to 1.31 mM. Average accuracy was 99.9±4.0% and the intra- and inter-day error of assay did not exceed 11%.     

Production and recovery of recombinant protease inhibitor α1-antitrypsin

Human α₁-antitrypsin (AAT) was produced in the recombinant yeast Saccharomyces cerevisiae ATCC 20699 grown in batch and fed-batch culture. The final biomass concentration and antitrypsin concentration attained were 55 g·L⁻¹ and 1.23 g·L⁻¹, respectively, in the fed-batch. The maximum productivities of biomass and antitrypsin were 1.6 and > 0.04 g L⁻¹h⁻¹, respectively, or substantially greater than the highest productivity values reported in the past. For recovering the antitrypsin, the cell slurry was concentrated 4-fold (231 g·L⁻¹ biomass, 122 min of processing) by cross-flow microfiltration and the cells were disrupted by bead milling (3 passes of 3 min total retention time). The cell homogenate was treated with aluminum chloride or PBS (pH 7) to aid separation of the cell debris by flocculation and sedimentation. The clarified cell homogenate was subjected to ammonium sulfate fractionation to precipitate the recombinant antitrypsin. The AAT precipitated at 45–75% saturation of ammonium sulfate, depending on the age of the homogenate. The crude AAT in the homogenate degraded at room temperature (25°C), with a zero order deactivation rate of 1.815 × 10⁻³ ± 3.43 × 10⁻⁴ g AAT L⁻¹h⁻¹.     

Oxygen isotope fractionation between human phosphate and water revisited

The oxygen isotope composition of human phosphatic tissues (δ¹⁸O_P) has great potential for reconstructing climate and population migration, but this technique has not been applied to early human evolution. To facilitate this application we analyzed δ¹⁸O_P values of modern human teeth collected at 12 sites located at latitudes ranging from 4°N to 70°N together with the corresponding oxygen composition of tap waters (δ¹⁸O_W) from these areas. In addition, the δ¹⁸O of some raw and boiled foods were determined and simple mass balance calculations were performed to investigate the impact of solid food consumption on the oxygen isotope composition of the total ingested water (drinking water + solid food water). The results, along with those from three, smaller published data sets, can be considered as random estimates of a unique δ¹⁸O_W/δ¹⁸O_P linear relationship: δ¹⁸O_W = 1.54(±0.09) × δ¹⁸O_P−33.72(±1.51) (R² = 0.87: p [H₀:R² = 0] = 2 × 10⁻¹⁹). The δ¹⁸O of cooked food is higher than that of the drinking water. As a consequence, in a modern diet the δ¹⁸O of ingested water is +1.05 to 1.2‰ higher than that of drinking water in the area. In meat-dominated and cereal-free diets, which may have been the diets of some of our early ancestors, the shift is a little higher and the application of the regression equation would slightly overestimate δ¹⁸O_W in these cases.     

Process for biological oxidation and control of dissolved iron in bioleach liquors

Iron has a central role in bioleaching and biooxidation processes. Fe²⁺ produced in the dissolution of sulfidic minerals is re-oxidized to Fe³⁺ mostly by biological action in acid bioleaching processes. To control the concentration of iron in solution, it is important to precipitate the excess as part of the process circuit. In this study, a bioprocess was developed based on a fluidized-bed reactor (FBR) for Fe²⁺ oxidation coupled with a gravity settler for precipitative removal of ferric iron. Biological iron oxidation and partial removal of iron by precipitation from a barren heap leaching solution was optimized in relation to the performance and retention time (τ_FBR) of the FBR. The biofilm in the FBR was dominated by Leptospirillum ferriphilum and “Ferromicrobium acidiphilum.” The FBR was operated at pH 2.0 ± 0.2 and at 37 °C. The feed was a barren leach solution following metal recovery, with all iron in the ferrous form. 98–99% of the Fe²⁺ in the barren heap leaching solution was oxidized in the FBR at loading rates below 10 g Fe²⁺/L h (τ_FBR of 1 h). The optimal performance with the oxidation rate of 8.2 g Fe²⁺/L h was achieved at τ_FBR of 1 h. Below the τ_FBR of 1 h the oxygen mass transfer from air to liquid limited the iron oxidation rate. The precipitation of ferric iron ranged from 5% to 40%. The concurrent Fe²⁺ oxidation and partial precipitative iron removal was maximized at τ_FBR of 1.5 h, with Fe²⁺ oxidation rate of 5.1 g Fe²⁺/L h and Fe³⁺ precipitation rate of 25 mg Fe³⁺/L h, which corresponded to 37% iron removal. The precipitates had good settling properties as indicated by the sludge volume indices of 3–15 mL/g but this step needs additional characterization of the properties of the solids and optimization to maximize the precipitation and to manage sludge disposal.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

The response of intact Strongyloides ratti infective (L3) larvae to substrates and inhibitors of respiratory electron transport

Live, intact third-stage larvae (L3s) of Strongyloides ratti in the absence of exogenous substrates consumed oxygen at a rate (E-QO₂) of 181.8 ± 12.4 ng atoms min⁻¹ mg dry weight⁻¹ at 35°C. Respiratory electron transport (RET) Complex I inhibitor rotenone (2 μm) produced 33 ± 6.5% inhibition of the E-QO₂. Unusually the rotenone-induced inhibition was not relieved by 5 μm-succinate. The E-QO₂ of intact L3s was refractory to RET Complex III inhibitor antimycin A at 2 μm; 4 μm-antimycin inhibited ≤ 10% of the E-QO₂. The electron donor couple ascorbate/TMPD augmented the E-QO₂ in the presence of rotenone (2 μm) and antimycin A (4 μm) by 110%. Azide (1 mm) stimulated the antimycin A refractory QO₂ by 36.6 ± 7.2% which was only partially inhibited by 1.0 mm-KCN (IC₅₀ = 0.8 mm). The data suggest the presence of classical (CPW) and alternate (APW) electron transport pathways in S. ratti L3s.     

Chromosomal aberrations in a fish, Channa punctata after in vivo exposure to three heavy metals

The studies were designed to assess the extent of chromosomal aberrations (CA) under the exposure of three common heavy metalic compounds, viz. mercuric chloride, arsenic trioxide and copper sulphate pentahydrate, in vivo using fish, Channa punctata (2n = 32), as a test model. Prior acclimatized fishes were divided into five groups. Group I and II served as negative and positive control, respectively. An intramuscular injection of Mitomycin-C (@ 1 mg/kg body wt.) was administered to group II only. Fishes of groups III, IV and V were subjected to sublethal concentrations (10% of 96 h LC₅₀), of HgCl₂ (0.081 mg/L), As₂O₃ (6.936 mg/L) and CuSO₄·5H₂O (0.407 mg/L). Fishes of all the groups were exposed uninterrupted for 24, 48, 72, 96 and 168 h. Observations of kidney cells of exposed fishes revealed chromatid and chromosome breaks, chromatid and chromosome gaps along with ring and di-centric chromosomes. A significant increase over negative control in the frequency of chromosomal aberrations (CA) was observed in fish exposed to Mitomycin-C, Hg(II), As(III) and Cu(II). As the average ± SE total number of CA, average number of CA per metaphase and %incidence of aberrant cells in Hg(II) was 104.40 ± 8.189, 0.347 ± 0.027 and 10.220 ± 0.842, respectively; in As(III) 109.20 ± 8.309, 0.363 ± 0.027 and 10.820 ± 2.347, respectively and in Cu(II) 89.00 ± 19.066, 0.297 ± 0.028 and 8.900 ± 0.853, respectively. Hence, it reveals that the order of induction of frequency of CA was Cu < Hg < As. The findings depict genotoxic potential of these metals even in sublethal concentrations.     

Cyclodextrin production by Bacillus firmus strain 37 immobilized on inorganic matrices and alginate gel

Production of β-cyclodextrin (β-CD) by Bacillus firmus strain 37 cells, immobilized by adsorption on silica–titania (SiO₂/TiO₂) and silica–manganese dioxide (SiO₂/MnO₂) matrices, was optimized for temperature, substrate concentration and initial biomass. The immobilization process was most efficient at 60 °C with 10% maltodextrin and 1.0 g of cells, resulting, after a 5-day assay, in a β-CD production of 11.7 ± 0.1 mM for cells immobilized on SiO₂/TiO₂ and 11.2 ± 0.1 mM in SiO₂/MnO₂. Entrapment in alginate gel resulted in a maximum β-CD production of 4.1 ± 0.1 mM, which was maintained constantly until the end of a 10-day assay. During this same period, free cells produced 8.3 ± 0.2 mM, and cells immobilized on SiO₂/TiO₂ and SiO₂/MnO₂, 16.7 ± 0.4 and 17.3 ± 0.5 mM, respectively. β-CD production by cells immobilized in calcium alginate in four repetitive cycles of 5 days each, showed an increase up to the third cycle, reaching 4.8 ± 0.2 mM, while production by free cells started falling from the second cycle. In this same assay, cells immobilized on SiO₂/TiO₂ and SiO₂/MnO₂, showed the best β-CD production results at the end of the first cycle, with a gradual fall occurring due to the desorption of cells thereafter.