Utilization of enzyme markers to determine the location of plasma membrane from Pisum epicotyls on sucrose gradients

Using linear sucrose gradients, particulates derived from pea (Pisum sativum L. cv. Alaska) epicotyls have been fractionated and examined for marker enzyme activity. The coincidence of three reputed plasma-membrane markers [cellulase (EC 3.2.1.4), K⁺-stimulated Mg²⁺-ATPase, and glucan synthetase] at the same position on sucrose density gradients, in combination with electron microscopic evidence reported by G. Shore and G. Maclachlan (J. Cell Biol. 64, 557–571; 1975), indicates that plasma membrane of pea epicotyl has a buoyant density of about 1.13 g/cm³. This density disagrees with those usually reported for plant plasma membranes and also with recent reports for Pisum. It is, however, shown to be distinct from the equilibrium densities of enzymic markers for particulate components derived from Pisum endoplasmic reticulum (1.10–1.11 g/cm³), Golgi (1.12 g/cm³) and mitochondria (1.18 g/cm³). Furthermore, other recent literature indicates that the 1.13 g/cm³ buoyant density may be characteristic of the plasma membrane of many members of the Leguminosae. Our data indicate that the conditions of differential centrifugation (time, centrifugal force), coupled with the amount of protein utilized, affect the resolution and interpretation of profiles of marker enzymes on sucrose gradients (e.g. glucan synthetase and K⁺-stimulated Mg²⁺-ATPase were sometimes found to be associated not only with particles of 1.13 g/cm³ density, but with particles of higher densities as well). Particulate cellulase was found to be associated only with particles with equilibrium densities of about 1.13 g/cm³. Cellulase thus proved to be the most useful marker for establishing a differential centrifugation regime which would permit examination of the 1.13 g/cm³ particulate components with minimal contamination by particles of higher densities.     

Mechanisms of Cl− uptake through brush border membranes isolated from the posterior intestine of the freshwater trout,Oncorhynchus mykiss,R.

Summary This paper presents a study of the mechanisms of Cl^– transport through the brush border membranes of the posterior part of the intestine in the freshwater trout, Oncorhynchus mykiss. The mechanisms for Cl^– transport in the posterior intestine are distinct from those in the middle intestine; an inwardly directed pH gradient stimulates Cl^– uptake by bursh border membrane vesicles, indicating a Cl^–/OH^– exchange. A pH-regulated Cl^– conductance is present, which is not activated at normal intracellular pH. Cl^– uptake is stimulated by an outwardly directed HCO ₃ ^– gradient revealing the presence of a Cl^–/HCO ₃ ^– exchange but, conversely, Cl^– is not exchanged against SO ₄ ^2- . In addition, carbonic anhydrase activities have been detected in both the intracellular and extracellular leaflets of the bursh border membranes which favour the establishment of a bicarbonate gradient. A model of Cl^– transport mechanisms through the brush-border membranes of the posterior intestine of the freshwater trout is proposed.Abbreviations BBM brush border membrane - CA carbonic anhydrase - EGTA ethylene-bis(oxyethylenenitrilo)tetra-acetic acid - FW fresh water - Hepes N-2-hydroxy-ethyl-piperazine-N'-2-ethanesulphonic acid - Mes 2-(N-morpholino)ethane sulphonic acid - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid - TEA triethanolamine - TMA tetramethylammonium - TRIS tris(hydroxymethyl)aminomethane     

Renal handling of taurine,l-alanine,l-glutamate andd-glucose inOpsanus tau: studies on isolated brush border membrane vesicles

Summary Renal brush border membrane vesicles (bbmv) from the aglomerular toadfish (Opsanus tau), isolated by differential precipitation, were tested for their ability to actively translocate (i) taurine, known to be secreted by the kidney of several marine teleosts, and (ii)l-alanine,l-glutamic acid, andd-glucose, solutes that are normally reabsorbed in the filtering nephron. Vesicular taurine uptake displayed a Na⁺ dependence. Transport was greatest under conditions of an inward-directed Na⁺ gradient, but a significant stimulation by Na⁺ over K⁺ could also be observed in the absence of a salt gradient. At high extravesicular K⁺, the addition of valinomycin reduced taurine uptake. Na⁺-dependent³H-taurine flux was almost completely inhibited by non-labeled taurine (tracer replacement) or -alanine, but was unaffected byl-alanine. Replacement of medium chloride by SCN^– or NO ₃ ^– in the presence of Na⁺ resulted in significantly lower uptake rates under both anion gradient and anion equilibrium conditions, whereas Br^– could almost fully substitute for the stimulatory Cl^– action. These results indicate the presence of an electrogenic Na⁺-cotransport mechanism with specificity for -amino acids in the toadfish renal brush border. Whether the system under physiological conditions mediates reabsorption or secretion of taurine remains to be determined. Toadfish bbmv also translocatedl-alanine andl-glutamic acid in a Na⁺-dependent manner. Possible roles for these most likely reabsorptive transport systems in a non-filtering kidney are discussed.d-glucose uptake, however, appeared to occur via Na⁺-independent pathways, since it was not affected by phlorizin in the presence of Na⁺, or by Na⁺ replacement.Abbreviation bbmv brush border membrane vesicles     

Passive H+/OH− permeability in epithelial brush border membranes

Passive H⁺/OH^– permeability across epithelial cell membranes is rapid and leads to partial dissipation of H⁺/OH^– gradients produced by H⁺ pumps and ion gradient-coupled H⁺/OH^– transporters. A heterogeneous set of H⁺/OH^– transport mechanisms exist in biological membranes: lipid solubility/diffusion, protein-mediated transport by specific proteins or by slippage through ion-coupled H⁺/OH^– transporters, and transport at the protein/lipid interface or through protein-dependent defects in the lipid structure. A variety of methods are available to study protein transport mechanisms accurately in cells and biomembrane vesicles including pH electrode recordings, pH-sensitive fluorescent and magnetic resonance probes, and potentiometric probes. In brush border vesicles from the renal proximal tubule, the characteristics of passive H⁺/OH^– permeability are quite similar to those reported for passive H⁺/OH^– permeability through pure lipid bilayers; slippage of protons through the brush border Na⁺/H⁺ antiporter or through brush border water channels is minimal. In contrast, passive H⁺/OH^– permeability in brush border vesicles from human placenta is mediated in part by a stilbene-sensitive membrane protein. To demonstrate the physiological significance of passive renal brush border H⁺/OH^– transport, proximal tubule acidification and cell pH regulation mechanisms are modeled mathematically for states of normal and altered H⁺/OH^– permeabilities.     

Use of Percoll™ in the isolation and purification of rabbit small intestinal brush border membranes

(1) Intestinal absorption is altered under a variety of circumstances in health and disease and to determine a possible relationship between intestinal absorptive function and intestinal brush border membrane composition, we undertook the isolation and purification of rabbit jejunal and ileal brush borders, to allow further studies of their lipid composition under varied experimental conditions. (2) A modification of an established method (Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J. and Crane, R.K. (1973) Biochim. Biophys. Acta 323, 98–112) utilized CaCl₂ aggregation and sequential centrifugation followed by purification of the brush border pellet (P₂) at 27 000 × g on a Percoll™ (Pharmacia) self-forming gradient. The Percoll™ was removed by ultracentrifugation for 30 min at 100 000 × g, utilizing a batch rotor in the Beckman airfuge™. (3) Pure brush border membrane vesicles were obtained and characterized by specific marker analysis and electron microscopy. Comparative marker analyses performed on P₂ and final Percoll™ preparations from animals showed that the purification achieved was 8–11-fold greater when compared to the original homogenates. Verification of purity was also demonstrated by the absence of DNA and very low levels of β-gluconridase and (Na⁺ + K⁺)-ATPase in the Percoll™ preparations. (4) Comparative lipid analyses of P₂ and final Percoll™ preparations showed that levels of total phospholipid and free fatty acids were several-fold higher in the Percoll™ preparations on a per mg protein basis. (5) A comparison of the activity of enzyme markers and the levels of total free fatty acids in P₂ pellets obtained after CaCl₂ and MgCl₂ aggregation showed that CaCl₂ aggregation gave the more consistently reproducible results. (6) Although standard procedures of membrane preparations not involving density gradient separation provide membranes of reasonable purity for the estimation of lipid components, we consider the final purification step of density gradient separation using Percoll™ is essential for determining small quantitative changes which might occur in the membrane lipid composition under experimental conditions where intestinal absorptive function is altered.     

Diffusion of sucrose in xanthan gum solutions

Molecular diffusion of solutes, like sucrose in the xanthan gum fermentation, is important in order to understand the complex behavior of mass transfer mechanisms during the process. This work was focused to determine the diffusion coefficient of sucrose, a carbon source for xanthan production, using similar sucrose and xanthan concentrations to those occurring in a typical fermentation. The diaphragm cell method was used in experimental determinations. The data showed that diffusion coefficient of sucrose significantly decreases when xanthan gum concentration increases. Theoretical and semiempirical models were used to predict sucrose diffusivity in xanthan solutions. Molecular properties and rheological behavior of the system were considered in the modeling. The models tested fitted well the behavior of experimental data and that reported for oxygen in the same system.List of Symbols A constant in eq. (5) - C _pg cm^–3 polymer concentration - D cm² s^–1 diffusivity - D _ABcm² s^–1 diffusivity of A through liquid solvent - D _APcm² s^–1 diffusivity of A in polymer solution - D _AWcm² s^–1 diffusivity of A in water - D _Pcm² s^–1 diffusivity of polymer in liquid solvent - E _D gradient of the activation energy for diffusion - H _P hydratation factor of the polymer in water (g of bound water/g of polymer) - K dyn sⁿ cm^–2 consistency index - K ₁ constant in eq. (5) - K _P overall binding coefficient [g of bound solute/cm³ of solution]/[g of free solute/cm³ of polymer free solution] - n flow behavior index - M _Bg g mol^–1 molucular weight of liquid solvent - M _Pg g mol^–1 molecular weight of the polymer - M _Sg g mol^–1 Molecular weight of polymer solution (= M _BX_B+M_PX_P) - R cm³ atm g mol^–1 K^–1 ideal gas law constant - T K absolute temperature - V _Bcm³ g mol^–1 molar volume of liquid solvent - V _Pcm³ g mol^–1 molar volume of polymer - V _Scm³ g mol^–1 molar volume of polymer solution - X _B solvent molar fraction - X _P polymer molar fraction - polymer blockage shape factor - _P volume fraction of polymer in polymer solution - g cm^–1 s^–1 viscosity - _ag cm^–1 s^–1 apparent viscosity of the polymer solution - _icm³ g^–1 intrinsic viscosity - ₀ g cm^–1 s^–1 solvent viscosity - _Pg cm^–1 s^–1 polymer solution viscosity - _R relative viscosity (= / ₀) - ₌₀ g cm^–1 s^–1 viscosity of polymer solution obtained at zero shear rate - ₀ g cm^–3 water density     

Prednisolone enhances aminopeptidase turnover in adult rat small intestine

In adult male rats, fed prednisolone (0.75 mg/kg/day) for 7 days, brush border aminopeptidase activity was increased (P < 0.001) by 106% compared to pair-fed controls. [¹⁴C]Tyrosine was injected intraperitoneally 16 h and [³H]tyrosine 6 h before death. The ³H/¹⁴C ratio was 1.79 ± 0.21 (S.D.) in purified microvillus membranes from treated rats compared to 1.30 ± 0.16 (P < 0.01) in controls. Polyacrylamide gel electrophoresis of brush border membranes under denaturing conditions showed that the increased double-isotope ratio in membranes from treated rats was mainly in the high molecular weight protein subunits (> 80 kDa) Detergent-solubilized aminopeptidase was purified after in vivo labeling by protein A-Sepharose-antiaminopeptidase affinity chromatography. The ³H/¹⁴C ratio in aminopeptidase was 2.42 ± 0.15 (P < 0.05) in treated rats compared to 1.63 ± 0.13 in controls. Over the experimental period steady-state isotope reutilization and protein labeling was demonstrated and there was no isotope metabolism. Total microvillus membrane lipid content was unaffected by prednisolone. We conclude that prednisolone increases brush border aminopeptidase activity by increasing enzyme turnover. Other high molecular weight brush border proteins were similarly affected.     

Density- and sound speed contrasts in sub-Arctic zooplankton

Summary The sound speed was determined for Meganyctiphanes norvegica, for a mixture of Thysanoessa raschii and Thysanoessa inermis and for a mixture of Calanus finmarchicus and Calanus hyperboreus. The sound speed contrasts ranged from 1.014 to 1.044. Seasonal variations in specific density were measured for Thysanoessa inermis, Thysanoessa raschii, Meganyctiphanes norvegica, Calanus finmarchicus and Calanus hyperboreus. The density of 20 mm T. inermis was lowest in November (1.052 g/cm³) and highest in February–March (1.065 g/cm³). For a 20 mm T. raschii the minimal density was determined in December (1.059 g/cm³) and the maximum in February–March (1.074 g/cm³). M. norvegica individuals of 35 mm also had their lowest density in December (1.060 g/cm³), but reached their maximum density in July (1.076 g/cm³).The density of the euphausiids was found to be size dependent. The density increases as the size decreases. C. finmarchicus and C. hyperboreus had densities less than seawater (1.026 g/cm³) during most of the year. Just before spawning the density increased to 1.028 g/cm³ and 1.036 g/cm³ for C. finmarchicus and C. hyperboreus respectively. The seasonal variations of the density were closely related to the lipid content of the animals.     

Two AT-rich satellite DNAs in the chironomid Glyptotendipes barbipes (Staeger)

Two AT-rich satellite DNAs are present in the genome of Glyptotendipes barbipes. The two satellites have densities of 1.680 g/cm³ (=21% GC) and of 1.673 g/cm³ (=13% GC) in neutral CsCl-density gradients. The main band DNA has a density of 1.691 g/cm³ (=32% GC). This value is in agreement with the 33% GC-content of G. barbipes DNA calculated from thermal denaturation (T_M=83° C). — In brain DNA as well as in salivary gland DNA the two satellite sequences together comprise 12–15% of the total G. barbipes DNA. Comparisons of the density profiles of DNA extracted from polytene and non-polytene larval tissue gave no hints for underreplication of the satellite DNAs during polytenization. — The two satellite DNAs have been isolated from total DNA by Hoechst 33258-CsCl density centrifugation and then localized in the polytene salivary gland chromosomes by in situ hybridization. Both satellite sequences hybridize to all heterochromatic centromere bands of all four chromosomes of G. barbipes. Satellite I (1.673 g/cm³) hybridizes mainly with the middle of the heterochromatin, satellite II (1.680 g/cm³) hybridizes with two bands at the margin of the heterochromatin. In situ hybridization with polytene chromosomes of Chironomus thummi revealed the presence of G. barbipes satellite sequences also in the Ch. thummi genome at various locations, mainly the centromere regions.     

Beneficial effects and mechanism of action of Momordica charantia juice in the treatment of streptozotocin-induced diabetes mellitus in rat

This study investigated the beneficial effects and mechanism of action of the juice of Momordica charantia in streptozotocin (STZ)-induced diabetes mellitus in rats. Diabetes mellitus was associated with significant (p < 0.01) time course reductions in body weight, plasma insulin and the number of insulin positive cells per islet and significant (p < 0.01) time course elevation in blood glucose and osmolarity and systolic blood pressure compared to age-matched healthy controls. Oral intake of M. charantia juice by STZ-induced diabetic rats partially reversed all the diabetes-induced effects measured. Daily oral administration of M. charantia juice to STZ-induced diabetic rates significantly (p < 0.01) reduced the Na⁺- and K⁺ -dependent absorptions of glucose by the brush border membrane vesicles of the jejunum compared to the responses obtained in STZ-induced diabetic rat. Either insulin (100 MM) or the fruit juice lyophilised extract (5 g · ml^–1) can stimulate ¹⁴C-D-glucose uptake in L6 myotubes. These effects were completely blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. High concentrations (10–200 g · ml^-1) of M. charantia juice extract inhibited ¹⁴C-D-glucose uptake in L6 myotubes compared to the control response. The effect of M. charantia treatment was also investigated on myelinated fibre abnormalities in the tibial nerve of STZ-induced diabetic and control rats. The results show that diabetes was associated with significant (p < 0.05) reduction in the mean cross-sectional myelinated nerve fibres, axonal area, myelin area and maximal fibre area compared to end controls. Treatment of STZ-induced diabetic rats with M. charantia juice normalised the structural abnormalities of peripheral nerves. The results indicate that M. charantia can exert marked beneficial effects in diabetic rats, and moreover, it can regulate glucose uptake into jejunum membrane brush border vesicles and stimulate glucose uptake into skeletal muscle cells similar to the response obtained with insulin. (Mol Cell Biochem 261: 63–70, 2004)     

Indigogenic methods for glycosidases

Summary 4-Cl-5-Br-3-indolyl--D-fucoside (IbF) was tested in histochemical and bio-chemical experiments. Sections from differently treated parts of the small intestine of suckling and adult rats, of jejunum of adult hamsters, guinea pigs, cooks, monkeys, of human jejunal biopsies, of kidney of suckling and adult rats, adult monkeys, guinea pigs and hamsters, and of some other rat organs were used in histochemical experiments. Neutral and acid -D-galactosidases prepared from homogenates of the small intestine of suckling rats by chromatography on Sephadex G 200 were used in biochemical experiments.The recommended medium for histochemical studies consists of 0.1 M citrate phosphate buffer pH 6 (brush border enzyme) and pH 4 (lysosomal enzyme), 3.6·10^–4M IbF and 3.1·10^–3 M potassium ferri- and ferrocyanide.IbF is split quite efficiently by the brush border -D-galactosidase (=lactase) and the recommended histochemical method with IbF at pH 6 is the method of choice in studies concerned with the localization of intestinal lactase. In unfixed cold microtome sections the brush border activity can be easily detected within 15–240 minutes, depending on the lactase activity of the studied sample. In this study this activity was shown to be present in the brush border of enterocytes in the upper part of crypts reaching its maximum in differentiated enterocytes covering the sides and tops of villi in the small intestine of suckling (the highest activity) and adult rats (3–4x lower activity according to the time of appearance of the staining), and of human, monkey and hamster jejunum. In the cock jejunum traces of brush border staining were seen only after 24 hours of incubation. In enterocytes of patients with celiac sprue the brush border activity was very much reduced in dependence on the stage of the disease. Brush border staining along with a diffuse cytoplasmic reaction was found in the proximal convoluted tubules of the monkey kidney. The question of localization of enzyme activity splitting IbF in the monkey kidney deserves further investigation.IbF is also split by isolated intestinal acid -D-galactosidase and histochemically a positive reaction was found in lysosomes of many cells displaying a high activity of -D-galactosidase when IbF was used in the recommended medium of pH 4. The use of aldehyde fixation (glutaraldehyde is to be preferred) is a prerequisite for the assessment of this lysosomal localization. The lysosomal activity splitting IbF is not firmly bound structurally and escapes from cold microtome sections prepared from unfixed tissue samples into the incubation solution.The recommended method is also very suitable for processing zymograms and immunoprecipitation lines of lactase with antisera obtained by Ouchterlony's technic and by immunoelectrophoresis.     

Effect of limited trypsin digestion on the renal Na+−H+ exchanger and its regulation by cAMP-Dependent protein kinase

Summary The Na⁺–H⁺ exchanger from solubilized rabbit renal brush border membranes is inhibited by cAMP-dependent protein kinase (PKA) mediated protein phosphorylation. To characterize this inhibitory response and its sensitivity to limited proteolysis, the activity of the transporter was assayed after reconstitution of the proteins into artificial lipid vesicles. Limited trypsin digestion increased the basal rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake in reconstituted proteoliposomes and blocked the inhibitory response to PKA-mediated protein phosphorylation. To determine if the inhibitory response to PKA-mediated protein phosphorylation could be restored to the trypsin-treated solubilized proteins, nontrypsinized solubilized brush border membrane proteins were separated by column chromatography. The addition of small molecular weight polypeptides, fractionated on Superose-12 FPLC (V _e=0.7), to trypsinized solubilized brush border membrane proteins restored the inhibitory response to PKA-mediated protein phosphorylation. Similarly, the addition of the 0.1m NaCl fraction from an anion exchange column, Mono Q-FPLC, also restored the inhibitory response to PKA. Both protein fractions contained a common 42–43 kDa protein which was preferentially phosphorylated by PKA.These results indicate that limited trypsin digestion dissociates the activity of the renal Na⁺–H⁺ exchanger from its regulation by PKA. It is suggested that trypsin cleaves an inhibitory component of the transporter and that this component is the site of PKA-mediated regulation. Phosphoprotein analysis of fractions that restored PKA regulation raises the possibility that a polypeptide of 42–43 kDa is involved in the inhibition of the renal Na⁺–H⁺ exchanger by PKA-mediated, protein phosphorylation.     

Visualization of lactotransferrin brush-border receptors by ligand-blotting

The uptake of iron (III) mediated by lactotransferrin to human biopsies from upper intestine has suggested the presence of specific receptors for human lactotransferrin at the brush border (Cox, T., Mazurier, J., Spik, G., Montreuil, J. and Peters, T.J. (1979) Biochim. Biophys. Acta 588, 120–128). In the present data, using ¹²⁵I-radiolabeled transferrins, we have demonstrated that a preparation of microvillous membrane vesicles, from rabbit jejunal brush-border specifically binds human lactotransferrin. This binding is specific, saturable and calcium dependent. Scatchard plots analysis of lactotransferrin binding indicates 1.5 · 10¹³ sites per mg of membrane proteins with an equilibrium constant of 1.2 · 10⁶ M⁻¹. Sodium dodecyl sulfate solubilization of the brush-border proteins allows the lactotransferrin receptor to retain its binding activity. Moreover, the ligand blotting of the detergent solubilized membrane proteins on nitrocellulose sheet and after incubation with ¹²⁵I-labeled lactotransferrin, has shown that the receptor is a protein of about 100 kDa. In the same experimental conditions, the rabbit microvillous membrane vesicles do not specifically bind rabbit serotransferrin indicating the absence of serotransferrin receptors at the brush border.     

Preparative electrophoresis: on the estimation of maximum temperature

The quantity of proteins processed by an electrophoretic technique is proportional to the cross-sectional area of the gel. For preparative purifications, an increase in the cross-sectional area is desired, but the Joule heating phenomenon restricts such an increase. The governing heat equation is analyzed and simplified with reference to Counteracting Chromatographic Electrophoresis. The application of the method of weighted residuals yields a compact and accurate solution for the maximum temperature rise in the column which is suitable for design calculations. Similar estimations indicate the efficiency of heat dissipation in annular configuration.List of Symbols C _p specific heat capacity, J g^–1 K^–1 - h heat transfer coefficient at the wall, W cm^–2K^–1 - i current density, A cm^–2 - k effective thermal conductivity of the packing, W cm^–1 K^–1 - k _b electrical conductivity of the buffer, mho cm^–1 - k _e effective electrical conductivity of the packing, mho cm^–1 - k _g electrical conductivity of the gel, mho cm^–1 - L length of the packing, cm - N _Pr Prandtl number - N _Re Reynolds number - r radial coordinate, cm - r _i inner radius of annulus, cm - r _o outer radius of annulus, cm - S heat source term, defined by eqn. (6) - T temperature, K - T _c cooling fluid temperature, K - T _i initial temperature, K - T _max highest temperature in the column, K - u superficial buffer velocity, cm s^–1 - V voltage gradient, V cm^–1 - porosity of the packing, dimensionless - buffer density, g cm^–3 - temperature, dimensionless Material presented in this paper has been adapted from the author's dissertation [15] which was accepted (supervisor: Dr. Jean B. Hunter) by the Cornell University Graduate Faculty in partial requirement of a graduate degree. Thoughtful discussions with Professors J. Robert Cooke and Michael L. Shuler regarding the annulus problem and the financial support provided by the Department of Agricultural and Biological Engineering, Cornell University, Ithaca, USA are gratefully appreciated.     

The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes

The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied. Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis. Phosphorylation was performed either in vivo by perfusion of the jejunum with [³²P]orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by [γ-³²P]ATP in the presence of saponin. The addition of cholera toxin (10 μg/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern. On the other hand, cAMP (at 5 μmol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by [γ-³²P]ATP. However, the same effect could also be induced by low concentrations of cGMP (0.1 μmol/l). It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.     

Lanthanide/proline cotransport across rabbit renal brush borders

Summary It has been suggested previously that La³⁺ can replace Na⁺ on various cotransport systems in renal brush border membranes. In the present study, we used rabbit renal brush border membrane vesicles to examine the specificity and kinetics of Ln³⁺/proline cotransport. Experiments were carried out under zero-trans, voltage clamped conditions using a rapid-mix/filtration technique. Initial experiments confirmed that La³⁺ produced the classical overshoot phenomenon. The initial rates of proline uptake relative to Na⁺ were Eu³⁺, Tb³⁺, Nd³⁺, Pr³⁺, Ho³⁺ (3.3)>Na⁺ (1.0)>La³⁺ (0.86) > choline⁺ (0.1). At a saturating salt concentration, uptake saturated with increasing proline concentration: theK _t andJ _max were 0.05mm and 17 pmol mg^–1 sec^–1 in Na⁺; and 0.28mm and 73 pmol mg^–1 sec^–1 in Tb³⁺. The higherJ _max in Tb³⁺ indicates that the Tb³⁺-proline loaded carrier is more effective than the Na⁺-proline loaded carrier in overcoming some rate-limiting barriers in the transport process. Na⁺ activated proline uptake with a Hill coefficient of 1.6 and aK _0.5 of 21mm, while Tb³⁺ activated with a Hill coefficient of 0.88 and aK _0.5 of 28mm. The Hill coefficient for Na⁺ suggests two binding sites, whereas the Hill coefficient for Tb³⁺ may indicate negative cooperativity between the trivalent ligands at the binding sites. We conclude that lanthanides are able to substitute for Na⁺ on the brush border proline carrier and that the lanthanides may serve as useful probes for the ligand binding sites.     

High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families

We have examined high affinity interactions of chick brain microtubule proteins with ³⁵S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1–4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65–75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. ³⁵S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM^–1 E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three ³⁵S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720–1.740 g/cm³; 1.750–1.765 g/cm³). These families are never observed as distinct bands in total DNA CsCl gradients, nor could we isolate them in purified tubulin control binding experiments. This apparently general phenomena may be identifying some of the sequence families involved in the high affinity microtubule interaction, which appears to be conserved in evolution.     

Laboratory culture of Chironomus tentans for use in toxicity testing: optimum initial egg-stocking densities

The midge Chironomus tentans Fabricius is a commonly used freshwater invertebrate in sediment toxicity tests. Rigorous laboratory culturing techniques are needed to provide organisms of uniform quality and known age for use in testing and for the continuation of the culture itself. This study was conducted to determine the effect of initial culture stocking density on: (1) post-hatch (larval) dry weight, body length and head-capsule width at 10 and 20 days; (2) time to emergence; (3) number and sex of emergent adults; (4) number of larvae and pupae at test termination (day 42 post hatch); and (5) adult dry weight. Three egg stocking densities were used 690 (1.1 eggs cm^–2), 1043 (1.7 eggs cm^–2) and 1463 (2.4 eggs cm^–2). Mean weight of larvae at 10 days in high density tanks (0.13 mg/organism) was significantly higher (P=0.003) than both the medium and low density tanks (0.10 and 0.09 mg/organism, respectively). No significant differences between the three stocking densities were observed for the body length or head-capsule width at either 10 or 20 days post-hatch. Although not statistically significant, larval dry weight decreased with increased stocking density at day 20. A significantly (P=0.02) greater number of females (173±28) emerged from the low stocking density compared to both the medium and high stocking densities (123±45 and 118±54, respectively). Peak adult emergence for the low and medium stocking densities occurred between days 22 and 25 post-hatch, whereas peak adult emergence occurred between days 30 and 33 for the high stocking density. Survival relative to the initial number of eggs stocked was significantly greater (P=0.007) in the low density treatment compared to that in either the medium or the high density treatments. Mean adult weight exhibited an inverse relationship with initial stocking densities. At test end, there was not a significant difference in the mean number of organisms surviving and emerging in the three density levels. The central tendency for number of organisms surviving for all three treatments was 504 organisms per tank (0.82 organisms cm^–2). The results of this experiment suggest that an optimal egg stocking density of 1.0 egg cm^–2 (600 eggs/tank) be used with the feeding rate identified. This would ensure uniform larvae at the appropriate developmental stage (2nd–3rd instar) needed for toxicological research/testing (e.g. 10 days post-hatch), as well as producing sufficient emergence of males and females for future culture establishment.     

The kinetics of anion equilibrium exchange across the red blood cell membrane as measured by means of35S thiocyanate

Summary Up to a SCN^– concentration of about 110mm, the concentration dependence of SCN^– equilibrium exchange in human red cell ghosts can be represented by the superimposition of two flux components. One component shows saturation kinetics, the other does not. The saturable component has an activation enthalpy of 105 kJ/mole, exhibits arans acceleration by Cl^– and can be inhibited by H₂DIDS. The nonsaturable component has a much lower activation enthalpy of 33 kJ/mole, is slightly reduced intrans acceleration experiments with Cl^– and insensitive to H₂DIDS but susceptible to inhibition by phloretin. At SCN^– concentrations exceeding 110mm, the saturable component undergoes irreversible self inhibition while the nonsaturable component remains unaltered.The half saturation concentration of the saturable flux component increases with decreasing pH from 3.0mm at pH 7.4 to 13.3mm at pH 6.0. Over this pH range, the maximal flux is only slightly increased from 19×10^–12 to 22×10^–12 moles×cm^–2×sec^–1. The nonsaturable flux component also increases slightly.In accordance with previous observations of Wieth (J. Physiol. (London) 207:563–580, 1970), we find that SCN^– increases K⁺ and Na⁺ permeability. The induced cation-permeability is considerably smaller than the SCN^– exchange and the latter does not show the paradoxical temperature dependence that is known to pertain to the former.     

Effects of nutrient (N,P, C) enrichment,grazing and depth upon littoral periphyton of a softwater lake

Three field experiments were performed in Lake Lacawac, PA to determine the importance of potentially limiting nutrients relative to other factors (grazing, depth) in structuring shallow water algal periphyton communities. All three experiments measured periphyton growth (as chlorophyll-a, AFDM or biovolumes of the algal taxa) on artificial clay flower pot substrates which released specified nutrients to their outer surfaces.Control of standing crop by nutrient supply rate vs. grazing was examined in Expt. I. Substrates releasing excess N and P, together with one of 4 levels of C (as bicarbonate) were placed either inside or outside exclosures designed to reduce grazer densities. Chlorophyll-a rose from 1.1–25.6 µg.cm^–2, and some dominant taxa (e.g., Oedogonium, Nostoc, Anacystis) were replaced by others (e.g., Scenedesmus, Cryptomonas) as bicarbonate supply increased. Reductions in invertebrate density did not significantly affect chlorophyll-a at any of the nutrient levels.Reasons for the species shift were further evaluated in Expt. II, using a minielectrode to measure the elevation of pH within the periphyton mat through photosynthetic utilization of bicarbonate. The pH adjacent to pots diffusing N, P and large quantities of bicarbonate, and supporting high chlorophyll-a densities of 32 µg cm^–2, averaged 10.0 compared to 6.3 in the water column. Pots diffusing only N and P supported 0.7 µg chlorophyll-a cm^–2 and elevated pH to 8.2. We suspect that bicarbonate addition favored efficient bicarbonate users (e.g., Scenedesmus), while inhibiting other taxa (e.g., Oedogonium) because of the attendant high pH.Expt. III was designed to test effects of depth (0.1 m vs. 0.5 m) and N (NH₄ ⁺ vs. NO₃ ^–) upon the growth response to bicarbonate observed in Expts. I and II. Similar standing crop and species composition were noted on pots at 0.1 m vs. 0.5 m. Enrichment with NH₄ ⁺ vs. NO₃ ^– also appeared to have little effect upon the periphyton community.Shallow water periphyton communities in Lake Lacawac, when supplied with sufficient N and P, appear to show a distinctive response to increasing bicarbonate concentration and pH which is robust to moderate variation in grazer densities, distance from the water surface, and the form of N enrichment.