Uptake of sewage derived nitrogen by Ulva rigida (Chlorophyceae) in Bahía Nueva (Golfo Nuevo, Patagonia, Argentine)

Uptake kinetics of nitrogen derived from sewage–seawater mixtures (2.5–20% v/v effluent) were determined in the laboratory for Ulva rigida (Chlorophyceae) native from Bahía Nueva (Golfo Nuevo, Patagonia, Argentine). In terms of nitrogen concentration, experimental enrichment levels varied between 53.7 and 362.3M of ammonium and between 0.77 and 6.21M of nitrate+nitrite. Uptake rates were fitted to the Michaelis–Menten equation, with the following kinetic parameters: ammonium: V_max = 591.2molg^–1h^–1, K _s=262.3M, nitrate+nitrite: V _max=12.9molg^–1h^–1, K _s=3.5M). Both nutrients were taken up simultaneously, but ammonium incorporation was faster in all cases. The results show a high capability of Ulva rigida to remove sewage-derived nitrogen from culture media. In the field, most of the nitrogen provided by the effluent would be tied up in algal biomass, supporting low nitrogen levels found at a short distance away from the source.     

The effects of pyridine nucleotides on the activity of a calcium-activated nonselective cation channel in the rat insulinoma cell line,CRI-G1

The activity of a calcium-activated nonselective (Ca-NS⁺) channel in a rat insulinoma cell line (CRI-G1) is inhibited by pyridine nucleotides in excised patches. The effects of all four pyridine nucleotides tested, -NAD⁺, -NADH, -NADP⁺ and -NADPH were very similar when tested at 0.1 mm, and at 1 mm the phosphorylated forms, -NADP⁺ and -NADPH, appeared to be slightly more potent than -NAD⁺ and -NADH. All the pyridine nucleotides tested reduced both the open state probability of the channel and the number of functional channels observed in a single patch.The application of -NAD⁺, but not of the other nucleotides tested, to the cytoplasmic surface of isolated inside-out patches from CRI-G1 cells opened a novel nonselective cation channel (the -NAD⁺-NS⁺ channel). The activity of this new channel is calcium sensitive and may also be inhibited by AMP.     

Electrochemical potential of protons in vesicles reconstituted from purified,proton-translocating adenosine triphosphatase

Summary Measurements were made of the difference in the electrochemical potential of protons ( ) across the membrane of vesicles reconstituted from the ATPase complex (TF ₀ ·F ₁) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential () and pH difference across the membrane ( pH), respectively.In the presence of Tris buffer the maximal and no pH were produced, while in the presence of the permeant anion NO ₃ ^– the maximal pH and a low were produced by the addition of ATP. When the ATP concentration was 0.24mm, the was 140–150 mV (positive inside) in Tris buffer, and the pH was 2.9–3.5 units (acidic inside) in the presence of NO ₃ ^– . Addition of a saturating amount of ATP produced somewhat larger and pH values, and the attained was about 310 mV.By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4–5 during ATP hydrolysis.The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

Isolation,purification and properties of tannase from Aspergillus niger van Tieghem

Tannase (tannin acyl hydrolase E.C. 3.1.1.20) has been isolated from Aspergillus niger van Tieghem and purified 29-fold. The enzyme had a temperature optimum of 60^°C, pH optimum of 6.0 with a second peak at pH 4.5, K_m of 0.20mM and V_max of 5.0mol min^–1 mg^–1protein.     

Conformational analysis of the Sda determinant-containing tetrasaccharide and two mimics in aqueous solution by using 1H NMR ROESY spectroscopy in combination with MD simulations

The conformational behaviour of the spacer-linked synthetic Sd^a tetrasaccharide -d-GalpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (1) and the two mimics -d-Galp-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (2) and -d-GlcpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (3) were investigated by ¹H NMR spectroscopy in combination with molecular dynamics (MD) simulations in water. Experimental 2D ¹H ROESY cross-peak intensities (ROEs) of the tetrasaccharides were compared with calculated ROEs derived from MD trajectories using the CROSREL program. Analysis of these data indicated that the oligosaccharidic skeletons of the compounds 1–3 are rather rigid, especially the -d-Hex(NAc)-(14)-[-Neu5Ac-(23)]--d-Galp fragments. The - Neu5-Ac-(23)--d-Galp linkage occurred in two different energy minima in the three-dimensional structure of the compounds 1–3 in aqueous solution. Experimental data and dynamics simulations supported the finding that the higher energy rotamer (CHEAT forcefield) was abundant in compounds 1 and 3 due to the existence of a hydrogen bond between the carboxyl group of the sialic acid and the acetamido group of the terminal monosaccharide (GalNAc or GlcNAc) unit. The conformational similarity between 1 and 3 leads to the suggestion that also their activities will be alike.     

N-andO-alkylation of glycoconjugates and polysaccharides by solid base in dimethyl sulphoxide/alkyl lodide

O-Methylation of simple neutral oligosaccharides is readily accomplished in dimethyl sulphoxide containing solid sodium hydroxide and methyl iodide [Cincanu I, Kerek F (1984) Carbohydr Res 131209-17]. This procedure has been extended to 2-acetamido-2-deoxy sugars and sialic acid-containing oligosaccharides. CompleteO-andN-methylation was in most cases achieved in 15 min. Esterification of carboxylic groups in uronic acids was fast and resulted in concomitant -elimination. The method is also suitable for methylation of glycoproteins and glycosphingolipids. Polysaccharides can also be methylated by this technique. Analysis of the products by gas-liquid chromatography and mass spectrometry showed no degradation products.Abbreviations lacto-N-tetraose LcOse₄, Gal3GlcNAc3Gal4Glc - lacto-N-fucopentaose III III³Fuc-nLcOse₄, Gal4[Fuc3]GlcNAc3Gal4Glc - trihexosylceramide GbOse₃Cer, Gal4Gal4Glc1-1Cer - globoside GbOse₄Cer, GalNAc3Gal4Glc1-1Cer - FAB-MS fas atom bombardment mass spectrometry     

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca²⁺ channel _1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although N-type Ca²⁺ channel plays a role in a variety of neuronal functions, _1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca²⁺ channel ₁ or subunit. In this study, we studied the levels of _1A, _1C, _1D, _1E, ₁, ₂, ₃ and ₄ mRNAs in adrenal gland of _1B-deficient mice. The _1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of _1C, _1D, _1E, ₁, ₂, ₃ and ₄ was found among wild, heterozygous and homozygous mice. The protein level of _1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of _1A gene, we examined lacZ expression in _1B-deficient × _1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of _1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous _1B-deficient × _1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of _1B-deficient mice is that compensation by _1A subunit occurs and that 6.3-kb 5-upstream region of _1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)     

“In situ” determinations of apparent K m and V max of brush border disaccharidases along the villi of normal human jejunal biopsy specimens

Summary In order to characterize lactase/-glucosidase and the neutral -glucosidase kinetically in the brush border membrane at different villus sites of normal human jejunal mucosa, a quantitative histochemical study has been carried out on biopsy specimens of patients only suffering from functional disturbances revealing normal mucosal architecture. The apparent V _max-values of both - and -glucosidases increased significantly from the villus base to the transition zone between medium and apical villus third. During enterocyte maturation on the villus no changes in the apparent K _m-values could be detected. Equally the ratios between the V _max-values of the disaccharidases remained constant. Male persons exhibited significantly greater apparent V _max-values for -glucosidase than females at both villus measuring positions. The findings can be taken as a starting point for the evaluation of local changes of apparent enzyme-kinetic data under pathological conditions.Supported by the Deutsche Forschungsgemeinschaft (GU 184/1)     

T-cell receptor V Tβ genes in natural populations of mice

The composition of 15 V _T gene subfamilies has been examined by Southern hybridization among a broad spectrum of colony bred rat and mouse species extending phylogenetically from Rattus to Mus musculus domesticus. Most mouse species contain a similar content of V _T genes as determined by the number of hybridizing restriction fragment (RF) bands. Furthermore, the extent of restriction fragment length polymorphism (RFLP) appears to be limited. Some V _T gene families, however, are missing from Rattus (VT7, V _T12) and M. shortridgei (V _T9, V _T16). Extension of the V _T survey to a panel of 38 wild-caught mice reveals that nearly a third lack specific hybridization to the V _T5 probe. Previous reports have established that the mouse inbred strains SJL, C57BR, C57L, and SWR lack 50% of their V _T repertoire, including V _T5 (Behlke et al. 1985). This study demonstrates that natural populations of mice also carry a significantly reduced V _T gene repertoire.     

A new mouse T-cell receptorα chain variable region family

Sequence analysis of the rearranged T-cell receptor a chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribedV gene family. We have designated this familyV 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously describedV haplotypes:V ^a ,V ^b , andV ^c .     

Linked genetic markers of the rabbit κ light chain are not linked to the Tcr β chain genes

In order to investigate linkage, we used serum allotypes of the two rabbit C isotypes and restriction fragment length polymorphisms (RFLPs) of the genes for V , C , and T-cell receptor C . The inheritance of these genetic markers was studied through backcross and F₂ matings. Southern analysis and hybridization of genomic DNA with a C probe detected a 5 kb Pst I fragment linked to expression of the K2bas1 allotype and the presence of the 1b ^bas gene and a 6.6 kb Pst I fragment linked to the expression of the K1b9 allotype, the presence of the 2 ^bas2 gene and lack of expression of the K2bas1 allotype. A V probe detected a 1.3 kb Eco RI fragment linked to the presence of the 1b ^bas gene and expression of the K2bas1 allotype. In contrast, the 9 or 14 kb Eco RI RFLP (C ^a or C ^b) detected with a Tcr chain probe segregated independently from C allotypes and RFLPs. It has previously been found that C and C are also unlinked in man, whereas in the mouse they are linked at a distance of 8 centimorgans.     

Light and GTP dependence of transducin solubility in retinal rods

The physical origin and functional significance of the near infra-red light scattering changes observable upon flash illumination of diluted suspensions of magnetically oriented, permeabilised frog retinal rods has been reinvestigated with particular attention paid to the degree with which transducin remains attached to the membrane. In the absence of GTP, the so called binding signal is shown to include two components of distinctive origins, widely different kinetics, and whose relative amplitudes depend on the dilution of the suspension and resulting detachment of transducin from the disc membrane. The fast component is a consequence of the fast interaction between photoexcited rhodopsin (R^*) and the transducin remaining on the membrane. Its kinetics monitors a structural modification of the discs caused by a change in electrostatic interaction between closely packed membranes upon the formation of R^*-T complexes. The slow component monitors the slow rebinding to the membrane and possible subsequent interaction with excess R^* of T-GDP which, in spite of its low solubility, had eluted into solution given the high dilution of the permeated rods. In the presence of GTP, the so called dissociation signal includes a fast, anisotropic release component that specifically monitors the release into the interdiscal space of T -GTP formed from the membrane-bound pool, and a slower isotropic loss component monitoring the leakage from the permeated rod of the excess T -GTP which did not interact with the cGMP phosphodiesterase. The amplitudes of both components depend exclusively on the membrane bound T-GDP pool. The kinetics of the loss component is limited by the size and degree of permeation of the rod fragments, rather than by the dissociation rate of T -GTP from the membrane.Abbreviations ROS rod outer segment - R rhodopsin - R^* photoactivated rhodopsin - T, T-GDP, T -GDP, T -GTP, T transducin and its various forms - T _mb, T _sol: T bound to membrane or soluble - PDE cGMP-phosphodiesterase - GTP guanosine 5-triphosphate - GDP guanosine 5-diphosphate - GDP S guanosine 5-O-(2-thiodiphosphate) - cGMP guanosine-3-5 cyclic-monophosphate - DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - TRIS Tris (hydroxymethyl)aminomethane - SDS sodium dodecyl sulfate     

\4 integrin expression onin vitro andin vivo metastatic variants of lewis lung carcinoma

The expression of 4, 6, and 1 integrin subunits has been investigated on somein vitro andin vivo murine metastatic variants derived from Lewis lung carcinoma (3LL). By the use of monoclonal antibodies which recognizes different epitopes of 6, 1, and 4 subunits we demonstrate that 6 and 1 subunits are expressed in all metastatic variants of 3LL irrespective of their metastatic potential, whereas 4 subunit is expressed only in highly metastasizing cells of 3LL. Northern blots of different metastatic variants probed with 1 and 4 subunits demonstrate thata) significant amounts of 1 mRNA were detected in all metastatic variants of 3LL;b) mRNA corresponding to the described entire coding sequence of 4 subunit is expressed only on highly metastasizing cells of 3LL. We conclude that 4 subunit is specifically expressed in highly metastasizig cells of 3LL while is undetectable in lower metastasizing ones.     

T-cell receptor-specific monoclonal antibodies against a V β 11-positive mouse T-cell clone

Two monoclonal antibodies specific for the mouse T-cell receptor (Tcr) have been established by immunization with a V 11⁺ T-cell clone, clone C6. One is a rat antibody, KT11 (IgG2b, k), specific for the V chain of C6, V 11. This was demonstrated by the fact that the strain distribution pattern of KT11⁺ cells was similar to that of V 5, 8, 9, 11, 12, and 13 and that the gene that encodes the molecule detected by KT11 was closely linked to V 8 in (B10 × SJL)F₁ × SJL backcross mice. Furthermore, V of C6 has been cloned from a gt10 cDNA library and was demonstrated to be identical to the V 11 published sequences. All strains of mice that do not express major histocompatibility complex class II E molecules had higher numbers of KT11^– cells than E⁺ strains. The KT11⁺ population in A strain mice and its H-2 congenic strains, however, was not affected by the presence or absence of E molecules. The other is a mouse antibody, KTL2 (IgM), specific for the idiotope of the Tcr expressed on the clone C6. Both antibodies were mitogenic and induced cytotoxicity. Expression of epitopes detected by KT11 or KTL2 was down-modulated by a T3-specific antibody 145-2C11.     

Inward rectifier K channels in renal epithelioid cells (MDCK) activated by serotonin

Summary The present study has been performed to test for the effect of intracellular calcium and of serotonin on the channel activity in patches from subconfluent MDCK-cells. In inside-out patches, inwardly rectifying potassium-selective channels are observed with open probabilities of 0.01±0.01, 0.24±0.03 and 0.39±0.07, at 100 nmol/liter, 1 mol/liter or 10 mol/liter calcium activity, respectively. The single-channel slope conductance is 34±2 pS, if the potential difference across the patch (V ) is zero, and approaches 59±1 pS, ifV is –50 mV, cell negative. In the cell-attached mode, little channel activity is observed prior to application of serotonin (open probability=0.03±0.03). If 1 mol/liter serotonin is added to the bath perfusate, the open probability increases rapidly to a peak value of 0.34±0.04 within 8 sec. In continued presence of the hormone, the open probability declines to approach 0.06±0.02 within 30 sec. At zero potential difference between pipette and reference in the bath (i.e., the potential difference across the patch is equal to the potential difference across the cell membrane), the single-channel conductance is 59±4 pS. In conclusion, inwardly rectifying potassium channels have been identified in the cell membrane of subconfluent MDCK-cells, which are activated to a similar extent by increase of intracellular calcium activity to 1 mol/liter and by extracellular application of 1 mol/liter serotonin.     

Eye specialisation in waterstriders: an adaptation to life in a flat world

The angular distribution of the optical axes of the ommatidia in the eyes of waterstriders (Gerris lacustris) has been investigated using antidromic illumination.

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet

Summary This communication reports the kinetics of the Na⁺/ Ca²⁺ exchanger and of the plasma membrane (PM) Ca²⁺ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca²⁺ extrusion and its Na⁺ dependence for concentrations of cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) in the 1–10-m range. The PM Ca²⁺ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca²⁺]_cyt] 1.5 m with the fluorescent Ca²⁺ indicator quin2 (K _d= 115 nm). That study determined that the PM Ca²⁺ pump in the basal state has a V _max = 0.098 mm/min, a K _m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca²⁺]_cyt with the intracellular Ca²⁺ probe, rhod2 (K _d= 500 nm), which has almost a fivefold lower affinity for Ca²⁺. An Appendix also describes the Mg²⁺ and pH dependence of the K _dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca²⁺ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca²⁺ extrusion into a Ca²⁺-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca²⁺ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca²⁺ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca²⁺ with a K _m= 0.97 ± 0.31 m and V_max = 1.0 ± 0.6 mm/ min. (ii) At high [Ca²⁺]_cyt, the exchanger works at a rate 10 times as large as the basal V _max of the PM Ca²⁺ extrusion pump. (iii) The exchanger can work in reverse after Na⁺ loading of the cytoplasm by monensin. (iv) The PM Ca²⁺ extrusion pump is activated by exposure to [Ca²⁺]_cyt 1.5 m for 20–50 sec. Activation raises the pump V _max to 1.6 ± 0.6 mm/min and the K _mto 0.55 ± 0.24 m. (v) The Ca²⁺ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca²⁺]_cyt. In summary, the results show that the human platelet can extrude Ca²⁺ very rapidly at high [Ca²⁺]_cyt. Both the Na⁺/Ca²⁺ exchanger and Ca²⁺ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca²⁺ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca²⁺]_cyt cytoplasmic Ca²⁺ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or V_extrusion true rate of Ca²⁺ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - _I fraction of high-affinity rhod2 complexed with Ca²⁺ - F the observed fluorescence - F_min the minimal fluorescence observed in the absence of Ca²⁺ - F_max the maximal fluorescence observed when the dye is saturated with Ca²⁺ - X₁ the fraction of high-affinity dye - K _d,1 dissociation constant of high-affinity dye - K _d,2 dissociation constant of the low-affinity dye - -d₁/dt rate of Ca²⁺ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - F_max (F_max — F_min)_cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F₅₀ fluorescence of the high-affinity form ofrhod2for[Ca²⁺]_cyt=50 nM - [Ca²⁺]₀ external Ca²⁺concentration - K _p proportionality constant between the total number of Ca²⁺ ions moved and the change in high-affinity rhod2 complexation to Ca² - (d[Ca²⁺]_{cyt, T})/dt rate of Ca²⁺ influx obtained with maximal levels of ionomycin - k_leak rate constant for passive inward Ca²⁺ leakage - k_inno rate constant for ionomycin-mediated Ca²⁺ influx - T total - [rhod2]_cyt,T total intracellular rhod2 concentration - [quin2]_cyt,T total intracellular quin2 concentration - [B]_T total cytoplasmic buffering capacity - A[Ca²⁺]_cyt,T total number of Ca²⁺ ions moved into the cytoplasm - [rhod2-Ca]_{cyt, T} change in concentration of total intracellular high-affinity rhod2 complexed to Ca²⁺ - [B-Ca]_T change in concentration of total cytoplasmic binding sites complexed to Ca²⁺ - [quin2]_{cyt, T} change in concentration of total intracellular quinl complexed to Ca²⁺ - change in the degree of intracellular quin2 saturation - ₁ change in degree of saturation of cytoplasmic high-affinity rhod2 - ₁-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - V_obs observed rate of Ca²⁺ removal from the rhod2-Ca complex - V_{8.3 m} the rate of Ca²⁺ removal from the high affinity rhod2-Ca complex at [Ca²⁺]_cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca²⁺]_cytT/_t initial linear rate of ionomycin-mediated Ca²⁺ influx - EC₅₀ effective concentration giving a half-maximal effect - [Na⁺]_cyt cytoplasmic Na⁺ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid