The Preparation of DNA Duplexes Containing Internucleotide Phosphorothioate Groups in Various Positions of the Recognition Site for the EcoRII Restriction Endonuclease

Twenty four 12-mer DNA duplexes, each containing a chiral phosphorothioate group successively replacing one of the internucleotide phosphate groups either in the EcoRII recognition site (5CCA/TGG) or near to it, were obtained for studying the interaction of the restriction endonuclease EcoRII with internucleotide DNA phosphates. Twelve of the 12-mer oligonucleotides were synthesized as R _p and S _p diastereomeric mixtures. Six of them were separated by reversed-phase HPLC using various buffers. Homogeneous diastereomers of the other oligonucleotides were obtained by enzymatic ligation of the R _p and S _p diastereomers of 5–7-mer oligonucleotides preliminarily separated by HPLC with the corresponding short oligonucleotides on a complementary DNA template.     

A nucleotide-independent nitroxide probe reports on site-specific stereomeric environment in DNA

In this report, stereospecific structural and dynamic features in DNA are studied using the site-directed spin labeling technique. A stable nitroxide radical, 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), was attached postsynthetically to phosphorothioates that were chemically introduced, one at a time, at five sites of a DNA duplex. The two phosphorothioate diastereomers (R_p or S_p) were separated, and nitroxide rotational motions were monitored using electron paramagnetic resonance spectroscopy. The resulting spectra vary according to diastereomer identity and location of the labeling site, with R_p-R5a spectra effectively reporting on local DNA structural features and S_p-R5a spectra sensing variations in local DNA motions. This establishes R_p- and S_p-R5a as unique probes for investigating nucleic acids in a site- and stereospecific manner, which may aid studies of stereospecific DNA/protein interactions. In addition, weighted averages of individual R_p and S_p spectra match those of R5a attached to mixed diastereomers. This suggests that R5a linked to mixed diastereomers reports on the composite behaviors of R_p- and S_p-R5a and is useful in initial probing of the DNA local environment. This work advances understanding of R5a/DNA coupling, and is a key step forward in developing a nucleotide-independent spectroscopic probe for studying nucleic acids.     

Protein recovery by continuous flotation

Summary Bovine serum albumin (BSA) was recovered from aqueous solutions by foam flotation. The protein concentrations in foam liquid C _S, in feed C _Pand in residue C _Rwere determined. The protein enrichment C _S/C_Pand the separation C _S/C_Ras well as the protein fraction in the foam liquid % BSA and foam liquid volume flow were determined as functions of the medium properties, operational conditions, and equipment parameters as well as concentrations of solid particles. At low protein concentrations in feed (e.g., C _P=40 mg · l^-1), and at 40° C, high performance was attained (C _X=2,000 mg · l^-1, C _R=4.4 mg · l^-1, C _S/C_P=50, C _S/C_R=450, 90% BSA. Continuous foam flotation is an efficient procedure for the recovery of low concentrations of proteins from liquid cultures.Abbreviations BSA bovine serum albumine - C _P protein concentration in feed (mg · l^-1) - C _R protein concentration in residue (mg · l^-1) - C _S protein concentration in foam liquid (mg · l^-1) - C _S/C_R protein separation (-) - C _S/C_P protein enrichment (-) - V _P feed rate (ml · min^-1) - V _R residue flow rate (ml · min^-1) - V _S foam liquid volume flow (ml · min^-1) - N number of theoretical stages in an ideal cascade (-) - temperature (° C) - mean residence time (min)     

Penicillin recovery from aqueous solutions by continuous foam flotation

Summary Penicillin G recovery is investigated in a continuous flotation column in the presence of different collectors which form a complex with penicillin. The performance of the penicillin recovery was investigated as a function of the mole ratio () of collector-to-penicillin and the aliphatic chain length of the collector. At =1 and low penicillin concentrations (e.g., 20 mg·1^-1), high foam liquid concentrations (680 mg·l^-1), low residue concentrations (12 mg·l^-1) and high penicillin separation (56) can be attained. At =4 the separation increases to 150, and 95% of the penicillin can be recovered.Symbols C_p penicillin concentration in feed (mg·l^-1) - C_R penicillin concentration in outlet liquid (mg·l^-1) - C_S penicillin concentration in foam liquid (mg·l^-1) - C_S/C_P penicillin enrichment (-) - C_S/C_R penicillin separation (-) - % Pen in S penicillin yield in foam liquid (%) - VV}_S foam liquid volume flow (ml·min^-1) - VV}_P feed (ml·min^-1) - VV_{N 2} nitrogen flow rate (ml·s^-1) - temperature     

A Molecular Dynamics Computer Simulation Study of Nucleotide Analogues. Comparison of the Hydration Pattern of Dithymidine Phosphate with those of the Dithymidine Methylphosphonate Diastereomers

Abstract

The hydration pattern of thymidyl(3′→5′) thymidine 1 and those of R_p and S_p diastereomers of the corresponding methylphosphonate analogue 2, have been studied using Molecular Dynamics (MD) computer simulation. It was found that the methylphosphonate modification leads to significant changes in the coordination of water molecules around the internucleotidic linkage and these, in turn, affect the hydration pattern of other parts of the molecule. The most notable differences between R_p and S_p diastereomers 2a and 2b were found to occur at the deoxyribose moieties of the nucleosid-5′-yl units.     

Comparison of the cleavage profiles of oligonucleotide duplexes with or without phosphorothioate linkages by using a chemical nuclease probe

A manganese porphyrin complex, Mn-TMPyP, associated with KHSO₅ is a chemical nuclease able to selectively recognize the minor groove of three consecutive AT base pairs of DNA and to mediate very precise cleavage chemistry at that particular site. This specific recognition and cleavage were used to probe the accessibility of the minor groove of DNA duplexes composed of one phosphodiester strand and one phosphorothioate strand. The cleavage of 5-GCAAAAGC/5-GCTTTTGC duplexes by Mn-TMPyP/KHSO₅ was monitored by HPLC coupled to electrospray mass analysis. Each single strand was synthesized with all-phosphate, all-Rp-phosphorothioate and all-Sp-phosphorothioate internucleotide bonds. We found that the manganese porphyrin was able to recognize its favorite (AT)₃-box binding site within the heteroduplexes, as in the case of natural DNA. Molecular modeling studies on the interactions of the reactive porphyrin manganese-oxo species with both types of duplexes confirmed the experimental data.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Syntheses and Biological Activities of (2S,2′ S)- and (2S,2′R)-Dimethyl 3-Phenyl-2,2′-oxydipropionate,Ether Analogues of a Plant-growth Regulator Isolated from Botrytis squamosa

Ether analogues of a plant growth regulator isolated from Botrytis squamosa, (2S,2′S)- and (2S,3′S)-dimethyl 3-phenyl-2,2′-oxydipro-pionate (1), were prepared by the Williamson synthesis, and the two diastereomers were separated. The absolute configurations of both diastereomers were determined by chemical transformation. The promoting effect of both synthesize analogues on lettuce seedling growth was similar to that of the natural plant growth regulator.     

Phosphate-limited growth of Chromatium vinosum in continuous culture

Chromatium vinosum DSM 185 was grown in continuous culture at a constant dilution rate of 0.071 h^-1 with sulfide as the only electron donor. The organism was subjected to conditions ranging from phosphate limitation (S _R-phosphate=2.7 M and S _R-sulfide=1.8 mM) to sulfide limitation (S _R-phosphate=86 M and S _R-sulfide=1.8 mM). At values of S _R-phosphate below 7.5 M the culture was washed out, whereas S _R-phosphate above this value resulted in steady states. The saturation constant (K ) for growth on phosphate was estimated to be between 2.6 and 4.1 M. The specific phosphorus content of the cells increased from 0.30 to 0.85 mol P mg^-1 protein with increasing S _R-phosphate. The specific rate of phosphate uptake increased with increasing S _R-phosphate, and displayed a non-hyperbolic saturation relationship with respect to the concentration of phosphate in the inflowing medium. Approximation of a hyperbolic saturation function yielded a maximum uptake rate (V _max) of 85 nmol P mg^-1 protein h^-1, and a saturation constant for uptake (K _t) of 0.7 M. When phosphate was supplied in excess 8.5% of the phosphate taken up by the cells was excreted as organic phosphorus at a specific rate of 8 nmol P mg^-1 protein h^-1.Non-standard abbreviations BChla bacteriochlorophyll a - D dilution rate; _max, maximum specific growth rate - maximum specific growth rate if the substrate were not inhibitory - K saturation constant for growth on phosphate - V _max maximum rate of phosphate uptake - K _i saturation constant for phosphate uptake - K _i inhibition constant for growth in the presence of sulfide - S _R concentration of substrate in the inflowing medium     

Evaluation of enantioselectivity in lipase-catalyzed acylation of hydroxyalkylphosphine oxides

The lipase-catalyzed optical resolution of 2-, 3-, and 5-hydroxyalkyl phosphorus compounds 1 provided the corresponding optically pure diastereomers in good yields. (S_P, R)- and (R_P, S)-1 were acylated faster than (S_P, S)- and (R_P, R)-1. The stereoselectivity at the phosphorus atom changed with the flexibility of the active sites in the lipases. The stereoselectivity at the phosphorus atom was higher in the reaction of 1a than in the reaction of 1b,c. The reaction rate of -hydroxyalkylphosphine oxide 1c was faster than that of 1a, although less enantioselectivity was observed at the phosphorus atom.     

Influence of pH,lactose and lactic acid on the growth of Streptococcus cremoris: a kinetic study

Summary The effect of various culture conditions on growth kinetics of an homofermentative strain of the lactic acid bacterium Streptococcus cremoris were investigated in batch cultures, in order to facilitate the production of this organism as a starter culture for the dairy industry. An optimal pH range of 6.3–6.9 was found and a lactose concentration of 37 g·l^-1 was shown to be sufficient to cover the energetic demand for biomass formation, using the recommended medium. The study of the effect of lactic acid concentration on growth kinetics revealed that the end-product was not the sole factor affecting growth. The strain was characterized for its tolerance towards lactic acid and a critical concentration of 70 g·l^-1 demonstrated. With the product yield of 0.9 g·g^-1 at non-lactose limiting conditions the lactic acid concentration of 33 g·l^-1 could not explain the low growth rates obtained, implicating a nutritional limitation.Symbols t _f fermentation duration (h) - X Biomass concentration (g·l^-1) - X _m maximum biomass concentration (g·l^-1) - S lactose concentration (g·l^-1) - S _r residual lactose concentration (g·l^-1) - P produced lactic acid concentration (g·l^-1) - P _a added lactic acid concentration (g·l^-1) - P _c critical lactic acid concentration (g·l^-1) - specific growth rate (h^-1) - _max maximum specific growth rate (h^-1) - R _x/S biomass yield (g·g^-1) calculated when =0 - R _P/S product yield (g·g^-1)     

Der Einfluß von Zwischenhirndefekten auf die Visuomotorik im Beute- und Fluchtverhalten der Erdkröte (Bufo bufo L.)

Zusammenfassung Eine Erdkröte (Bufo bufo L.), die sich in Beutestimmung befindet, antwortet auf ein kleines, in der Horizontalen um sie herum bewegtes visuelles Muster S _zmit orientierenden Wendereaktionen R _z, S_z R_z. Die Reaktivität im Zuwenden R _zist von verschiedenen Parametern des visuellen Reizes S _zabhängig: u. a. von der Winkelgeschwindigkeit, der Gestalt, der Flächengröße und dem Vorzeichen des Reiz-Hintergrund-Kontrastes. Die Winkelgeschwindigkeit. Für Sehwinkelgeschwindigkeiten von 30–60°/sec ist R _zmaximal; R _z=0, wenn die Geschwindigkeit=0 oder >200°/sec. Die Gestalt. Die Auslösewirkung einer Rechteckfläche ist von ihrer Horizontal-(h) und Vertikalausdehnung (v) abhängig. Verlängerung der Vertikalkante wirkt auf R _zgrundsätzlich hemmend, Verlängerung der Horizontalkante dagegen stimulierend. So wird R _zz.T. durch das Kantenlängenverhältnis h/v bestimmt: R _zsinkt, wenn h/v<1 und steigt, wenn h/v>1. Die Flächengröe. Für h/v=1 ist die Reaktivität R _zauf Flächen von h·v=4°·4° bis 8°·8° maximal. Wenn h·v>32°·32°, antworten die Kröten oft mit Bewegungen, die ein Abwenden R _avom Reiz S _azum Ziele haben, S _a R _a (Funktionskreis: Flucht). Die Reaktivität im Abwenden R _aist ebenfalls von der Bewegungsgeschwindigkeit und der Flächengröße des visuellen Musters abhängig. Das Vorzeichen des Reiz-Hintergrund-Kontrastes (weiße Fläche auf schwarzem Hintergrund oder umgekehrt). Bei schwarzen Flächen ist die Abhängigkeit der Reaktivität R _zvom Kantenlängenverhältnis h/v deutlicher ausgeprägt als bei weißen. Dementsprechend lösen weiße Flächen allgemein stärker aus als schwarze. Die Reaktivität im Abwenden R _aist dagegen auf schwarze Flächen sehr viel höher als auf weiße.Durch Erhöhung der Beutestimmung bei zusätzlicher olfaktorischer Reizung (Mehlwurmkotduft) wird die h/v- und h·v-abhängige Spezifität von R _zreduziert.Beutefangbewegungen (Zuwenden, Schnappen) lassen sich auch durch punktförmige elektrische Reizung des retinalen Projektionsfeldes im Tectum opticum auslösen. Fluchtbewegungen (Abwenden, Ducken, Springen) werden dagegen überwiegend durch elektrische Reizung der praetectalen Region oder des caudalen dorsalen Thalamus aktiviert.Nach Ausschaltung der praetectalen Region und des dorsalen Thalamus fällt das visuelle Fluchtverhalten aus; die Visuomotorik im Beutefang ist dagegen enthemmt. Die Tiere beantworten dann auch große visuelle Bewegungsmuster mit Beutefangreaktionen (S _a R_z), die vor der Operation Flucht ausgelöst hatten (S _a R_a). Unabhängig vom Verhältnis h/v steigt R _zmit zunehmender Flächengröße h·v an und nähert sich dabei einem Sättigungswert.Die Versuchsergebnisse geben weitere Einblicke in die Reizverarbeitung der Visuomotorik im Beute- und Fluchtverhalten der Anuren. Die verhaltensphysiologischen Ergebnisse werden mit neurophysiologischen Befunden an der Froschnetzhaut verglichen (Grüsser et al.). Der mutmaßliche Reiz-Reaktionsmechanismus wird diskutiert.

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The influence of diencephalic lesions on visually released movements in prey-catching and escape behaviour of the common toad (Bufo bufo L.)

Summary The prey-catching behaviour of Anura is released by small visual patterns. When toads (Bufo bufo L.) are about to catch food, their first action is turning movement (R _z) towards prey (S _z), visual stimulus pattern): S _z R_z. It was found that the following stimulus parameters determine the reactivity R _z: angular velocity, stimulus size, and direction of the contrast. Angular Velocity. For angular velocities of 30 to 60°/sec is R _zat a maximum. R _z=0 if the velocity=0 or >200°/sec (Fig. 3). Angular Size. Horizontally moved horizontal rectangular patterns have a greater releasing value than vertical rectangular patterns (Figs. 5A–7A). The releasing effect of a rectangular pattern depends on its horizontal h and vertical v expansion. On principle, an elongation of the vertical edge has inhibiting effects on R _z(Fig. 6A), an elongation of the horizontal edge, however, has stimulating effects (Fig. 5A). R _zpartly is determined by the proportion of the edge lengths h/v; R _zis decreasing if h/v}<1 and it is increasing if h/v>}1 (Fig. 8). For h/v=1 R _zis at a maximum, if h·v = 4°·4° to 8°·8° (Fig. 4A). If h·v}>32°·32° the toads often react by movements aiming at turning away (R _a) from the stimulation locality (S _a): S_a R_a (escape behaviour). The reactivity of turning away R _adepends as well on the angular velocity and the angular size of the visual pattern (Figs. 3 and 10). Direction of Contrast (white pattern on black background or vice versa). In case of black patterns (Figs. 4A, b to 7A, b) the reactivity R _zdepends to a far greater extent on the ratio h/v than in case of white patterns (Figs. 4A, a to 7A, a). Consequently white patterns have in general a greater releasing value than black ones. The reactivity of turning away R _a, however, is much stronger in case of black patterns than in case of white ones.In stimulation the prey-catching behaviour by motivational factors (additional olfactory stimulation) the characteristic depending on h/v is reduced (Fig. 9a and b).Prey-catching movements (turning towards prey, snapping) can be also released by electrical point stimulation of the projection area of the retina in the tectum opticum. Escape movements (turning to flight, crouching, jumping), however, are mainly activated by electrical stimulation of the pretectal region or the caudal dorsal thalamus (Fig. 13).After disturbing the pretectal region and the dorsal thalamus, TP-Iesion (Figs. 2, 11 and 12), escape behaviour fails, whereas the visually released movements in prey-catching behaviour are disinhibited (Fig. 10). The animals then respond also to great moving patterns by prey-catching reactions (S _a R _z), which before the operation would normally have caused escape (S _a R_a). Independent of the ratio h/v R _zis increasing with growing area h·v and approaching to a saturation value (Figs. 4B–7B).The data obtained in behavioural experiments are compared with findings of corresponding neurophysiological experiments with the frog's retina (Grüsser et al.). The presumable stimulus/reaction mechanism is discussed (Fig. 15).

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Mit Unterstützung der Deutschen Forschungsgemeinschaft Ew 7/1b, 7/2 und 7/4.

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Herrn Dr. O.-J. GRÜSSER (Dozent am Physiologischen Institut der Freien Universität Berlin) danke ich für kritische Durchsicht des Manuskripts.     

Constrained C-terminal hexapeptide neurotensin analogues containing a 3-oxoindolizidine skeleton

Summary In order to enforce different spatial orientations in the C-terminal hexapeptide of neurotensin (NT_8–13) and to gain information about the importance of the 10–11 peptide bond for binding to NT receptors, the Pro¹⁰-Tyr¹¹ fragment has been replaced with (2R,8S,8aR)-, (2S,8S,8aR)-, (2S,8S,8aS)-, (2S,8R,8aS)- and (2R,8R,8aS)-8-amino-2-benzyl-3-oxoindolizidine-2-carboxylic acid. Molecular dynamics calculations and energy minimization studies have shown that, contrarily to the Pro-Tyr moiety, none of these indolizidines display a tendency to adopt type I and III -turns, but those having (8S,8aR) or (8R,8aS) stereochemistry essentially adopt extended conformations and the (8S,8aS) stereoisomer prefers a nonstandard folding. The four diastereomeric NT_8–13 analogues incorporating (8S,8aR) or (8R,8aS) indolizidines displayed binding affinities for the brain NT receptor similar to that of [Ala¹¹]-NT_8–13 and only five- to ninefold lower than that of the corresponding analogue, [Phe¹¹]NT_8–13. Although this slight decrease could be attributed to differences in conformational behavior between these constrained NT_8–13 analogues and [Phe¹¹]NT_8–13 or NT_8–13, it is not clear whether the -turn around Pro¹⁰-AA¹¹ (AA=Phe, Tyr) is conserved upon receptor binding. An excessive restriction in the motions of the aromatic side chain, imposed by the highly steric constraint of the indolizidine moiety, emerges as an alternative explanation. The findings reported here demonstrate the possibility of replacing the Pro¹⁰-Tyr¹¹ dipeptide in NT_8–13 with a non-peptide residue without affecting considerably the affinity for brain NT receptors.     

Ventilatory responses to exercise and carbon dioxide in elderly and younger humans

The present investigation examined the relationship between CO₂ sensitivity [at rest (S _R) and during exercise (S _E)] and the ventilatory response to exercise in ten elderly (61–79 years) and ten younger (17–26 years) subjects. The gradient of the relationship between minute ventilation and CO₂ production ( _E/ CO₂) of the elderly subjects was greater than that of the younger subjects [mean (SEM); 32.8 (1.6) vs 27.3 (0.4); P<0.01]. At rest, S _R was lower for the elderly than for the younger group [10.77 (1.72) vs 16.95 (2.13) 1 · min^–1 · kPa^–1; 1.44 (0.23) vs 2.26 (0.28) 1 · min^–1 · mmHg^–1; P<0.05], but S _E was not significantly different between the two groups [17.85 (2.49) vs 19.17 (1.62) l · min^–1 · kPa^–1; 2.38 (0.33) vs 2.56 (0.21) 1 · min^–1 · mmHg^–1]. There were significant correlations between both S _R and S _E, and _E/ CO₂ (P<0.05; P<0.001) for the younger group, bot none for the elderly. The absence of a correlation for the elderly supports the suggestion that _E/ CO₂ is not an appropriate index of the ventilatory response to exercise for elderly humans.     

Stereoselective uptake of the GABA-transaminase inhibitors gamma-vinyl gaba and gamma-acetylenic GABA into neurons and astrocytes

The cellular uptake of the GABA-transaminase inhibitors gamma-vinyl GABA (GVG) and gamma-acetylenic GABA (GAG) was studied in cultured neurons and astrocytes. By the use of the individual enantiomersR- andS-GVG andR- andS-GAG it could be shown that in both cell types only theS-enantiomers could be actively transported. Comparing neurons and astrocytes only neurons exhibited a high affinity uptake system forS-GVG (K _m 78.2±20.3 M;V _max 0.71±0.06 nmol · min^–1 · mg^–1 cell protein). In case ofS-GAG it could not be established with certainty whether the neuronal uptake was of the high affinity type. Both GVG and GAG were studied as inhibitors of GABA uptake into neurons and astrocytes.S-GVG andS-GAG were found to be weak inhibitors of GABA uptake suggesting thatS-GVG is not transported by the GABA carrier in neurons. The finding of a much more efficient uptake ofS-GVG into neurons than into astrocytes is in line with the previous observation that neuronal GABA-T is more sensitive than astrocytic GABA-T toS-GVG.     

Stereoisomers of Mn(III) complexes of ethylenebis[(o-hydroxyphenyl)glycine]

We have prepared the Mn(III) complexes rac-Na[Mn(EHPG)]·3H₂O (1) and rac,meso-Na[Mn(EHPG)]·H₂O (2), where H₄EHPG is ethylenebis[(o-hydroxyphenyl)glycine], and determined their X-ray crystal structures. Complex 1 contains N(S,S)C(R,R) configurations at the N and C stereogenic centres, whilst in the unit cell of complex 2 there are two independent molecules, 2a (meso) and 2b (rac), with N(R,R)C(S,R) and N(R,R)C(S,S) configurations, respectively. Enantiomers of each complex are also present. The Mn(III) centres have Jahn-Teller-distorted octahedral geometry. The rac isomer has two long axial MnO(carboxylate) bonds (2.162-2.202 Å) and the equatorial plane contains two short MnN bonds (2.012-2.063 Å) trans to short MnO(phenolate) bonds (1.865-1.901 Å). The meso isomer has long axial MnN (2.194 Å) and MnO(carboxylate) (2.152 Å) bonds, and shorter equatorial MnN (2.005 Å) trans to MnO(phenolate) (1.901 Å) and MnO(carboxylate) (1.988 Å) trans to O(phenolate) (1.897 Å) bonds.     

Oligonucleotides containing a peptide internucleotide linkage. A novel class of modified oligonucleotides

Oligonucleotide analogues containing one or a few glycine, L-, and D-alanine residues instead of phosphodiester internucleotide linkages were synthesized (C3′-NH-C(O)-CH(X)-NH-C(O)-C4′, where X = H, (S)-CH₃, and (R)-CH₃. The stability of the duplexes of modified oligonucleotides with their wild-type complements was studied. The incorporation of glycine and L-alanine residues into internucleotide linkages was shown to noticeably decrease the stability of modified duplexes as compared to that of native ones (ΔT _m∼−2°C per modification), whereas analogues containing D-alanine linkers form duplexes with increased stability (ΔT _m∼+2°C per modification).     

Aldosterone regulates paracellular pathway resistance in rabbit distal colon

Summary Regulation of the paracellular pathway in rabbit distal colon by the hormone aldosterone was investigated in vitro in Ussing chambers by means of transepithelial and microelectrode techniques. To evaluate the cellular and paracellular resistances an equivalent circuit analysis was used. For the analysis the apical membrane resistance was altered using the antibiotic nystatin. Under control conditions two groups of epithelia were found, each clearly dependent on the light: dark regime. Low-transporting epithelia (LT) were observed in the morning and high-transporting epithelia (HT) in the afternoon. Na⁺ transport was about 3-fold higher in HT than in LT epithelia. Incubating epithelia of both groups with 0.1 mol·1^-1 aldosterone on the serosal side nearly doubled in LT epithelia the short circuit current and transepithelial voltage but the transepithelial resistance was not influenced. Maximal values were reached after 4–5 h of aldosterone treatment. In HT epithelia due to the effect of aldosterone all three transepithelial parameters remained constant over time. Evaluation of the paracellular resistance revealed a significant increase after aldosterone stimulation in both epithelial groups. This increase suggests that tight junctions might have been regulated by aldosterone. The hormonal effect on electrolyte transport was also dependent on the physiological state of the rabbit colon. Since net Na⁺ absorption in distal colon is, in addition to transcellular absorption capacity, also dependent on the permeability of the paracellular pathway, the regulation of tight junctions by aldosterone may be a potent mechanism for improving Na⁺ absorption during hormone-stimulated ion transport.Abbreviations V _t transepithelial potential difference (mV) - R _t transepithelial resistance (·cm²) - G _t transepithelial conductance (mS·cm^-2) - I_sc calculated short circuit current (A·cm^-2) - V _a apical membrane potential difference (mV) - V _bl basolateral membrane potential difference (mV) - voltage divider ratio - R _a apical membrane resistance (·cm²) - R _bl basolateral membrane resistance (·cm²) - R _c cellular resistance ( of apical and basolateral resistance) (·cm²) - R _p resistance of the paracellular pathway (·cm²) - G _a apical membrane conductance (mS·cm^-2) - G _bl basolateral membrane conductance (mS·cm^-2) - G _p paracellular conductance (mS·cm^-2) - G _t transepithelial conductance (mS·cm^-2) - HT _contr high transporting control epithelia - LT _contr low transporting control epithelia - HT _aldo aldosterone incubated high transporting epithelia - LT _aldo aldosterone incubated low transporting epithelia     

Ethanol production with Zymomonas mobilis with synthetic medium in batch operation

Ethanol was produced with Zymomonas mobilis Z6 (ATCC 29191), in batch culture with synthetic medium on glucose as substrate and in the presence of aspartate. The concentrations of glucose, phosphate, ammonium, ethanol and dissolved O₂ and CO₂ in the medium and O₂ and CO₂ in the outlet gas as well as the cell mass by culture fluorescence were measured on-line. Cell mass, glucose and aspartate concentrations were measured off-line. In the presence of a sufficient amount of aspartate, the ethanol inhibition effect can be reduced considerably. However, the improvement with yeast extract is more incisive. The relationship between the intensity of culture fluorescence and cell mass concentration is linear, if sufficient aspartate is present.List of Symbols ASP kg/m³ aspartate concentration - CTR kg/(m³ · h) CO₂ transfer rate - N, NH₄ kg/m³ nitrogen concentration from NH ₄ ⁺ - P kg/m³ product (ethanol) concentration - p% product (ethanol) yield - PO₄ kg/m³ phosphate concentration - Q _E kg/(kg · h) specific ethanol production rate - kg/(kg · h) specific nitrogen uptake rate from NH ₄ ⁺ - Q _P kg/(kg · h) specific phosphate uptake rate - Q _s kg/(kg · h) specific substrate (glucose) uptake rate - S kg/m³ glucose concentration - S _O kg/m³ initial glucose concentration - Y _x/s kg/kg yield coefficient - h^–1 specific growth rate     

Cell recovery by continuous flotation

Summary Cell recovery by means of continuous flotation of the Hansenula polymorpha cultivation medium without additives was investigated as a function of the cultivation conditions as well as of the flotation equipment construction and flotation operational parameters. The cell enrichment and separation is improved at high liquid residence times, high aeration rates, small bubble sizes, increasing height of the aerated column, and diameter of the foam column. Increasing cell age and cultivation with nitrogen limitation reduce the cell separation.Symbols C_P cell mass concentration in medium g·l^–1 - C_R cell mass concentration in residue g·l^–1 - C_S cell mass concentration in foam liquid g·l^–1 - V equilibrium foam volume cm³ - V gas flow rate through the aerated liquid column cm³·s^–1 - V_F feed rate to the flotation column ml/min - ¹ V _S/V foaminess s - mean liquid residence time in the column s     

Antiplatelet Activity,P2Y1 and P2Y12 Inhibition,and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate

Background

Diadenosine tetraphosphate (Ap₄A), a constituent of platelet dense granules, and its P¹,P⁴-dithio and/or P²,P³-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y₁ and P2Y₁₂. The most active of those analogs, diadenosine 5′,5″″-P¹,P⁴-dithio-P²,P³-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.

Objective

To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y₁ and P2Y₁₂ receptor antagonism, and their metabolism in human plasma.

Methods

We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y₁-mediated changes in cytosolic Ca²⁺, P2Y₁₂-mediated changes in VASP phosphorylation, and metabolism in human plasma.

Results

The inhibition of ADP-induced human platelet aggregation and human platelet P2Y₁₂ receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P¹- and P⁴-phosphorothioate groups, the S_PS_P diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the R_PR_P diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of S_PR_P diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P²,P³-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the S_PS_P and the R_PR_P diastereomers.

Conclusions

The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y₁ and P2Y₁₂ receptors.