Alanine and taurine transport by the gill epithelium of a marine bivalve: Effect of sodium on influx

Summary Marine mussels can accumulate amino acids from seawater into the epithelial cells of the gill against chemical gradients in excess of 5×10⁶ to 1. Uptake of both alanine and taurine into gill tissue isolated fromMytilus californianus was found to be dependent upon Na⁺ in the external solution. Uptake of these amino acids was described by Michaelis-Menten kinetics, and a reduction in external [Na⁺] (from 425 to 213mm) increased the apparent Michaelis constants (alanine, from 8 to 17 m; taurine, from 4 to 39 m) without a significant influence on theJ _max's of these processes. Fivemm harmaline, an inhibitor of Na-cotransport processes in many systems, reduced both alanine and taurine uptake by more than 95%; this inhibition appeared to be competitive in nature, with an apparentK _i of 43 m for the interaction with alanine uptake. Increasing the external [Na⁺] from 0 to 510mm produced a sigmoid activation of alanine and taurine uptake withK _Na's of approximately 325mm. The apparent Hill coefficients for this activation were 7.3 and 7.4 for alanine and taurine, respectively. These data are consistent with uptake mechanisms which require comparatively high concentrations of Na⁺ to activate transport, and which couple several Na⁺ ions to the transport of each amino acid. These characteristics, in conjunction with the previously demonstrated low passive permeability of the apical membrane to amino acids, result in systems capable of i) accumulating amino acids from seawater to help meet the nutritional needs of this animal, and ii) maintaining the high intracellular amino-acid concentrations associated with volume regulation in the gill.     

Single-channel K+ currents inDrosophila muscle and their pharmacological block

Summary Four types of nonvoltage-activated potassium channels in the body-wall muscles ofDrosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA). Ba²⁺, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The K_ST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130mm KCl solution), was blocked by TEA (K _d 35mm), and was the most sensitive to Ba²⁺ (complete block at 10^–4 m). A Ca²⁺-activated potassium channel. K_CF 72pS (130/130mm KCl), was gated open at>10^–8 m Ca²⁺, was the least sensitive to Ba²⁺ (K _d of 3mm) and TEA (K _d of 100mm), and was not affected by quinidine. K₂ was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1mm. It also exhibited a half block at 0.3mm Ba²⁺ and 25mm TEA. A fourth channel type, K₃, was the most sensitive to TEA (half block<1mm). It displayed a partial block to Ba²⁺ at 10mm, but no block by 0.1mm quinidine. The blocking effects of TEA, Ba²⁺ and quinidine were reversible in all channels studied. The actions of TEA and Ba²⁺ appeared qualitatively different: in all four channels. TEA reduced the apparent unitary conductance, whereas Ba²⁺ decreased channel open probability.     

Mutant of the glutamine transfer RNA gene as UGA suppressor in Escherichia coli

Summary A UGA suppressor derived from a glutamine tRNA gene of Escherichia coli K 12 was isolated and characterized. Phages carrying the suppressor su⁺2_UGA could be obtained only from a hybrid transducing phage, h ⁸⁰ cI ₈₅₇psu ⁺2_oc, but not from the original transducing phage cI ₈₅₇psu ⁺2_oc. By DNA sequence analysis, it was found that the su ⁺2 UGA suppressor obtained has two mutations; one is in the anticodon (TTATCA), as expected, and the other (CT) is at the 7th position from the 3 end of tRNA ₂ ^Gln . The significance of these mutations and the lethal effect on phage of the increased amounts of UGA suppressor tRNAs are discussed.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Effect of NaCl on kinetics ofd-glucosamine uptake in yeasts differing in halotolerance

The initial rate ofd-glucosamine uptake by the non-halotolerant yeastSaccharomyces cerevisiae was approximately halved as the apparent half saturation constant (K_m) and the apparent maximum velocity (V_max) changed from 6.6mm to 16.4mm and from 22 μmol · g⁻¹ · min⁻¹ to 16 μmol · g⁻¹ · min⁻¹, respectively, when the salinity in the medium was increased from zerom to 0.68m NaCl. Corresponding changes in a high affinity transport system in the halotolerant yeastDebaryomyces hansenii were from 1.1mm to 4.6mm and from 3.1 μmol · g⁻¹ · min⁻¹ to 4.5 μmol · g⁻¹ · min⁻¹, implying a practically unchanged transport capacity. In 2.7m NaCl, K_m and V_max in this system were 24.5mm and 1.1 μmol · g⁻¹ · min⁻¹, respectively, representing a marked decrease in transport capability. Nevertheless, the degree of affinity in this extreme salinity must still be regarded as noteworthy. In addition to the high affinity transport system inD. hansenii, a low affinity system, presumably without relevance ind-glucosamine transport, was observed.     

d-Alanine metabolism in the lucinid clam Lucinoma aequizonata

The chemoautotrophic symbiont-bearing clam Lucinoma aequizonata contains very high levels of free d-alanine in all tissues. The possible sources for this amino acid and its involvement in the clams' metabolism were investigated. Very low levels of d-alanine (generally below 1 mol·l^-1) were measured in the sediment porewaters from the habitat of the clams. Experiments with ¹⁴C-labeled tracers demonstrate an active metabolism of d-alanine in the clams rather than a role as inert waste product. d-alanine is metabolized at about 0.12 mol·g fw^-1·h^-1. Label from aspartate, but not glucose and CO₂, is incorporated into d-alanine. Incubation with labeled d-alanine did not result in formation of radioactive l-alanine. Tests for alanine racemase (EC 5.1.1.1) and d-amino acid oxidase (EC 1.4.3.3.) did not show activity in either gill, i.e. symbiont and host, or foot tissue. d-Alanine amino transferase (EC 2.6.1.b.) was demonstrated in gill and foot tissues. Two sources for d-alanine are proposed: a degradation of cell walls of symbiotic bacteria and production by the host using a d-specific alanine transaminase.Abbreviations aa amino acid(s) - fw fresh weight - HPLC high-performance liquid chromatography - MBH methyl benzethonium hydroxyde - NAC N-acetyl-l-cysteine - OPA ortho-phthaldialdehyde - TCA tricarbonic acid     

Ca2+-activated K+ conductance of the human red cell membrane: Voltage-dependent Na+ block of outward-going currents

Summary Human red cells were prepared with various cellular Na⁺ and K⁺ concentrations at a constant sum of 156mm. At maximal activation of the K⁺ conductance,g _K(Ca), the net efflux of K⁺ was determined as a function of the cellular Na⁺ and K⁺ concentrations and the membrane potential,V _m , at a fixed [K⁺]_ex of 3.5mm.V _m was only varied from (V _m –E _K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g _K(Ca) as a function of cellular Na⁺ and K⁺ concentrations atV _m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na⁺. Since the present Ca²⁺-activated K⁺ channels have been shown to be of the multi-ion type, the experimental data from each set of Na⁺ and K⁺ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na⁺ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na⁺ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca²⁺ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg _K(Ca) for outward currents at (V _m –E _K)>25 25 mV reflects a voltage-dependent Na⁺ block of the Ca²⁺-activated K⁺ channels.     

Diffusive water permeability in isolated kidney proximal tubular cells: Nature of the cellular water pathways

Summary The diffusive water permeability (P _d ) of the plasma membrane of proximal kidney tubule cells was measured using a¹H-NMR technique. The values obtained for the exchange time (T _ex) across the membrane were independent of the cytocrit and of the Mn²⁺ concentration (in the range 2.5 to 5mm). At 25°C the calculatedP _d value was (per cm² of outer surface area without taking into account membrane invaginations) 197±17 m/sec. This value equals 22.3±1.9 m/sec when the invaginations are taken into account. Cell exposure to 2.5mm parachloromercuribenzenesulfonic acid,pCMBS, (for 20 to 35 min) reducedP _d to 45% of its control value. Fivemm dithiothreitol, DTT, reverted this effect. The activation energy for the diffusive water flux was 5.2±1.0 kcal/mol under control conditions. It increased to 9.1±2.2 kcal/mol in the presence of 2.5mm pCMBS. Using our previous values for the osmotic water permeability (P _os) in proximal straight tubular cells theP _os/P _d ratio equals 18±1, under control conditions, and 3.2±0.3 in the presence ofpCMBS. These experimental results indicate the presence of pathways for water, formed by proteins, crossing these membranes, which are closed bypCMBS. Assuming laminar flow (within the pore), fromP _os/P _d of 13 to 18 an unreasonably large pore radius of 12 to 15 Å is calculated which would not hinder cell entry of known extracellular markers. Alternatively, for a single-file pore, 11 to 20 would be the number of water molecules which would be in tandem inside the pore. The water permeability remaining in the presence ofpCMBS indicates water permeation through the lipid bilayer. There are similarities between these results and those obtained in human red blood cells and in the apical cell membrane of the toad urinary bladder.     

Models of the bis-histidine-coordinated ferricytochromes: Mössbauer and EPR spectroscopic studies of low-spin iron(III) tetrapyrroles of various electronic ground states and axial ligand orientations

The EPR and magnetic Mössbauer spectra of a series of axial ligand complexes of tetrakis(2,6-dimethoxyphenyl)porphyrinatoiron(III), [(2,6-(OMe)₂)₄TPPFeL₂]⁺, where L=N-methylimidazole, 2-methylimidazole, or 4-(dimethylamino)pyridine, of one axial ligand complex of tetraphenylporphyrin, the bis(4-cyanopyridine) complex [TPPFe(4-CNPy)₂]⁺, and of one axial ligand complex of tetraphenylchlorin, [TPCFe(ImH)₂]⁺, where ImH=imidazole, have been investigated and compared to those of low-spin Fe(III) porphyrinates and ferriheme proteins reported in the literature. On the basis of this and previous complementary spectroscopic investigations, three types of complexes have been identified: those having (d_xy)²(d_xz,d_yz)³ electronic ground states with axial ligands aligned in perpendicular planes (Type I), those having (d_xy)²(d_xz,d_yz)³ electronic ground states with axial ligands aligned in parallel planes (Type II), and those having the novel (d_xz,d_yz)⁴(d_xy)¹ electronic ground state (Type III). A subset of the latter type, with planar axial ligands aligned parallel to each other or strong macrocycle asymmetry that yield rhombic EPR spectra, cannot be created using the porphyrinate ligand. Type I centers are characterized by "large g_max" EPR spectra with g>3.2 and well-resolved, widely spread magnetic Mössbauer spectra having A_zz/g_NN>680 kG, with A_xx negative in sign but much smaller in magnitude than A_zz, while Type II centers have well-resolved rhombic EPR spectra with g_zz=2.4–3.1 and also less-resolved magnetic Mössbauer spectra, and usually have A_zz/g_NN in the range of 440–660 kG (but in certain cases as small as 180 kG) and A_xx again negative in sign but only somewhat smaller (but occasionally larger in magnitude) than A_zz, and Type III centers have axial EPR spectra with g_┴2.6 or smaller and g_║<1.0–1.95, but often not resolved, and less-resolved magnetic Mössbauer spectra having A_zz/g_NN in the range of 270–400 kG, and A_xx again negative in sign but much smaller in magnitude than A_zz. An exception to this rule is [TPPFe(4-CNPy)₂]⁺, which has A_xx/g_NN=–565 kG, A_yy/g_NN=629 kG, and A_zz/g_NN=4 kG. A subset of Type II complexes (Type II) have rhombicities (V/) much greater than 0.67 and A_zz/g_NN ranging from 320 to 170 kG, with A_xx also negative but with the magnitude of A_xx significantly larger than that of A_zz. These classifications are also observed for a variety of ferriheme proteins, and they lead to linear correlations between A_zz and either A_xx, g_zz, or V/ for Types I and II (but not for A_zz versus V/ for Type II). Not enough data are yet available on Type III complexes to determine what, if any, correlations may be observed.Abbreviations CCP cytochrome c peroxidase - 4-CNPy 4-cyanopyridine - cyt cytochrome - EFG electric field gradient - ESEEM electron spin echo envelope modulation - ImH imidazole - Mb myoglobin - MCD magnetic circular dichroism - 2-MeImH 2-methylimidazole - N-MeIm N-methylimidazole - 3NH₂PzH 3-aminopyrazole - 4-NMe₂Py 4-(dimethylamino)pyridine - [2,6-(OMe)₂]₄TPP dianion of tetrakis(meso-2,6-dimethoxyphenyl)porphyrin - OEiBC dianion of octaethylisobacteriochlorin - OEP dianion of octaethylporphyrin - PPIX dianion of protoporphyrin IX - Py pyridine - TMP dianion of meso-tetramesitylporphyrin - TPC dianion of meso-tetraphenylchlorin - TPP dianion of meso-tetraphenylporphyrin - 2,6-XylylNC 2,6-xylyl isocyanide     

Growth of Juvenile Centropomus undecimalis in a Tropical Island

Age, growth rates and hatching dates were determined for juvenile common snook, Centropomus undecimalis, collected in several Puerto Rican estuarine systems. Two growth models (von Bertalanffy and power curve) were used and compared based on the fitting, the residual distribution and homogeneity of variances. A multiple linear regression was also fit to predict age from otolith weight, fish weight and standard length. Hatching dates back-calculated from age estimates, suggested that spawning occurs throughout the year but peaks from May to October. Early juveniles (age < 100d) recruited during the wet season (August to November) showed a significantly higher growth rate (0.67mmd^–1) than the dry season (December to July) recruits (0.41mmd^–1). These results are compared with snook collected in west Florida.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

The quantal gating charge of sodium channel inactivation

Using a very low noise voltage clamp technique it has been possible to record from the squid giant axon a slow component of gating current (I _g ) during the inactivation phase of the macroscopic sodium current (I _Na ) which was hitherto buried in the baseline noise. In order to examine whether this slowI _g contains gating charge that originates from transitions between the open (O) and the inactivated (I) states, which would indicate a true voltage dependence of inactivation, or whether other transitions contribute charge to slowI _g , a new model independent analysis termed isochronic plot analysis has been developed. From a direct correlation ofI _g and the time derivative of the sodium conductance dg _Na/d the condition when only O-I transitions occur is detected. Then the ratio of the two signals is constant and a straight line appears in an isochronic plot ofI _g vs. dg _Na/d . Its slope does not depend on voltage or time and corresponds to the quantal gating charge of the O-I transition (q _h ) divided by the single channel ionic conductance (). This condition was found at voltages above – 10 mV up to + 40 mV and a figure of 1.21e ^– was obtained forq _h at temperatures of 5 and 15°C. At lower voltages additional charge from other transitions, e.g. closed to open, is displaced during macroscopic inactivation. This means that conventional Eyring rate analysis of the inactivation time constant _h is only valid above – 10 mV and here the figure forq _h was confirmed also from this analysis. It is further shown that most of the present controversies surrounding the voltage dependence of inactivation can be clarified. The validity of the isochronic plot analysis has been confirmed using simulated gating and ionic currents.Abbreviations I _g gating current - I _Na sodium ionic current - g _Na macroscopic sodium conductance - single channel conductance - C, O, I closed, open, inactivated state occupancy of channels - g _h quantal charge displaced in a single O-I transition of Na channel - e ^– equivalent electron charge - h index referring to inactivation process - S _l limiting slope in isochronic plot see Eq.(3) - fractional distance, see Fig. 4 and (4, 5) - TMA tetramethylammonium - TTX tetrodotoxin - Tris tris(hydroxymethyl)aminomethane - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Light-temperature interactions on the growth of Antarctic diatoms

Summary The combined effect of various temperatures and light intensities on the growth of seven species of antarctic diatoms in culture has been studied. With the exception of Chaetoceros deflandrei whose thermal tolerance is fairly good, these obligatory psychrophils cannot survive in temperatures above 6° to 9° C. Their mean growth rate is relatively low, between 0.24 div d^–1 for Corethron criophilum and 0.63 div d^–1 for C. deflandrei. Regardless of light intensity, growth rate increased with the temperature to reach a maximum between 3° and 5° C. The highest rates were obtained between 115 and 220 mol m^–2 s^–1 with 0.38 div d^–1 for C. criophilum, 0.56 div d^–1 for Synedra sp. and between 0.71 and 0.88 div d^–1 for the other 5 species. A reduction in light intensity from 220 to 46 mol m^–2 s^–1 slowed growth by nearly 50%. These results suggest that the combined effect of temperature and light is one of the factors involved in the limitation of antarctic phytoplankton growth. The low temperatures of the environment do not permit rapid growth, which, even under optimal light conditions remains low. In addition, in the euphotic layer, the overall light energy available for algae is considerably reduced due to turbulence, a factor which exacerbates the reduced growth rate.     

Isolation of an abnormally phosphorylated erythrocyte membrane band 3 glycoprotein from patients with myotonic muscular dystrophy

Summary A fraction of erythrocyte Band 3 (M _r , 93,000) glycoprotein that demonstrates decreased autophosphorylation in membranes from myotonic muscular dystrophy patients is demonstrated. Sequential affinity chromatography of Triton X-100 solubilized erythrocyte membrane proteins separated three specifically retained glycoprotein fractions on a Ricin Communis I-Sepharose 4B column. One fraction contains a portion of the major sialoglycoprotein (apparentM _r , 78,000) and is specifically eluted from the column by 10mm NaCl and 100mm d-galactose (10/100). The two other glycoprotein fractions are eluted by 100mm NaCl, 10mm d-galactose (100/10) and 100mm NaCl, 100mm d-galactose (100/100). The composition of both fractions contains greater than 95% Band 3 (apparentM _r , 93,000) glycoprotein.The quantities of glycoprotein in each fraction obtained from erythrocytes of myotonic dystrophy patients did not differ from the quantities obtained from control erythrocytes. Following endogenous protein kinase incubations of ghosts with [-³²P]ATP, the specific [³²P] phosphorylation of the 10/100 and 100/10 fractions are identical. The 100/100 fraction, which makes up approximately 3% of the total erythrocyte membrane protein, demonstrates a different pattern for myotonic dystrophy patients; specific phosphorylation was reduced by 50% relative to activity in control experiments. These findings are consistent with previous experiments that demonstrated decreased autophosphorylation of the glycoprotein portion of Band 3 (Roses & Appel, 1975,J. Membrane Biol. 20: 51) and are consistent with the autosomal dominant mode of inheritance in this disease.     

Application of fuzzy multiobjective optimization for self-sucking impellers in a bioreactor

For three types of self-sucking impellers (fourand six-pipe and disk impellers) mixing power, initial point, amount of gas leaving the impeller and mass transfer coefficient were determined experimentally. Investigations were performed for two systems: water and biomass solution.From the point of view of a minimum mixing power and maximum mass transfer coefficient the best impeller has been chosen. Fuzzy multiobjective optimization for determination of optimum operating conditions is proposed.List of Symbols c concentration of oxygen - D tank diameter - d impeller diameter - g acceleration of gravity - H height of liquid in the tank - H height of liquid above impeller, H=H-y - k consistency coefficient - k _L a volumetric mass transfer coefficient - N rotational speed of impeller - n flow behaviour index - P mixing power for pure liquid - P _G mixing power for aerated liquid - V _G volumetric air flow rate - y distance of impeller from the tank bottom - v _a apparent kinematic viscosity of liquid - density of liquid - time - gas hold-up - Eu=P/N ³ d ⁵ or Eu_G=P _G /N ³ d ⁵ Euler Number for non-gassed or aerated liquid - Fr=N ² d/g Froude Number - Fr^*=N ² d ² /g(H -y) modified Froude Number - K_G=V _G /N d ³ gas flow number - Re=N d ² /v _a Reynolds Number - Sh=k _K a/(g ² /v _a )^1/3 Sherwood Number     

Use of a biosensor to determine the binding kinetics of five lectins for Galactosyl-N-acetylgalactosamine

The dietary lectins, edible mushroom (ABL) and Jacalin (JAC) inhibit the proliferation of colonic cancer cells, whereas Amaranth (ACL) and peanut (PNA) stimulate their proliferation. All these lectins share as their preferred ligand the Thomsen-Friedenreich (TF) antigen galactosyl 1,3 N-Acetylgalactosamine (Gal1,3GalNAc), but differ in their finer specificities for modifications of this determinant and in their specificities for cancerous epithelia. We have investigated, using a resonant mirror biosensor, the kinetics of binding of these lectins, and Maclura pomifera lectin (MPL), which is similar to JAC, to two different Gal-GalNac bearing glycoproteins, antarctic fish antifreeze glycoprotein (AFG) and asialofetuin. JAC had the highest affinity for AFG [K _d 0.027 M] due to a fast association rate constant [k _ass 610,000 (Ms)^–1]. The other lectins had considerably lower affinities, with K _d ranging from 0.16 M (ABL) to 5.7 M (PNA), largely due to slower k _ass [ABL 74,000 (Ms)^–1 to PNA 2700 (Ms)^–1]. Similarly, JAC had a much higher affinity for asialofetuin [K _d 0.083 M] than the other lectins [K _d 1.0 M–4.5 M]. Affinities were also calculated from the extent of binding at equlibrium and were generally similar to those calculated from the kinetic parameters indicating the true nature of these values.     

Morphological and Physiological Correlates of Evolutionary Reduction of Metabolic Rate among Amblyopsid Cave Fishes

I report on multiple correlates of reduction in metabolic rate during evolutionary adaptation to caves in the fish family Amblyopsidae. The family includes six species with one surface-dweller, Chologaster cornuta, a facultative cave dweller, Chologaster agassizi, and four obligate cave-dwellers that, on the basis of eye degeneration, have been isolated in caves for increasing times: Typhlichthys subterraneus Amblyopsis spelaea Amblyopsis rosae Speoplatyrhinus poulsoni. Of those traits I examined, the strongest correlates of reduction in whole fishes metabolic rate were reduction in ventilation frequency and volume > brain metabolic rate > gill surface area. Relative amplitude declined and the estimated ventilation minute-volume decreased 5.9 fold from a value of 1.12 to 0.19 ml. Log brain rate of oxygen consumption was directly related to log body mass, b = + 0.75, and decreased from 1.5 to 0.06 per mg dry mass per hour among species. No single gill trait accounted for the reduction in total lamellar surface area from 228 to 103 mm² g^–1. There was no reduction in muscle metabolic rate or histological indices of thyroid activity. Log muscle rate of oxygen consumption was inversely related to log body mass, b = –0.60, but did not differ among species. The mean total thyroid follicle volume among species, from 0.010 to 0.020 mm³ g^–1 of adult fishes, showed no relation to interspecific differences in whole fish metabolic rate. In the general discussion I show that no trait that could contribute to a lowered metabolic rate is in the same rank order as metabolic rate, though collectively the reductions were in the same order as metabolic rates. I explain that this is as expected with convergent evolution of complex traits. Finally I discuss the literature about central nervous system control of metabolic rate.     

Mixing performance of air-lift fermenters against working volume and draft tube dimensions

Mixing characteristics of a laboratory scale internal loop air-lift fermenter has been investigated. The effects of different draft tube dimensions and positions as well as varying levels of liquid height over the draft tube, on mixing time were determined. The results indicate the existance of an optimum liquid height and thus liquid volume with respect to mixing performance especially for the taller draft tubes.List of Symbols A mm distance between draft tube and reactor base - A _D mm² area of the downcomer region - A _R mm² area of the riser region - B mm width of annulus - D _d mm draft tube diameter - D _t mm fermenter diameter - H _d mm draft tube height - H _l mm liquid height in the fermenter - H _t mm fermenter height - V _d m³ draft tube volume - V _t m³ fermenter volume - D _d /D _l - _B H _d /H _l - _F H _l /D _t