Crystallization and preliminary X-ray analysis of phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus.

Recombinant phosphoserine aminotransferase (EC 2.6.1.52) from Bacillus circulans subsp. alkalophilus was crystallized at room temperature from 0.1 M sodium acetate buffer, pH 4.6, and 2% PEG 20000, using macroseeding techniques. The crystals diffract X-rays to at least 2.0 A nominal resolution. They belong to space group C2 with unit cell dimensions a = 93.2 A, b = 93.1 A, c = 45.6 A, alpha = 90.0 degrees, beta = 106.8 degrees, gamma = 90.0 degrees. A native data set to 2.3 A has been collected. Assuming an average packing density of the crystals, there is one monomer in the asymmetric unit, resulting in a calculated solvent content of 48.2%.     

The design and pharmacology of novel selective muscarinic agonists and antagonists

The muscarinic pharmacology of C1-methyl-substituted chiral compounds related to McN-A-343 and of (R)- and (S)-dimethindene has been studied. Among the McN-A-343 analogues, the (S)-enantiomers were more potent and had higher affinity than the (R)-isomers. The quaternary compound (S)-BN 228 was found to be the most potent M1-selective agonist known today (pEC50: M1/rabbit vas deferens = 7.83; M2/guinea-pig atria = 6.35; M3/guinea-pig ileum = 6.29). In both the atria and ileum the tertiary carbamate, (S)-4-F-MePyMcN, was a competitive antagonist (pA2 value = 7.39 and 6.82, respectively). In contrast, in rabbit vas deferens (S)-4-F-MePyMcN was a potent partial agonist (pEC50 = 7.22; apparent efficacy = 0.83). These results indicate that (S)-4-F-MePyMcN might be a useful tool to study M1 receptor-mediated effects involved in central cholinergic function. (S)-Dimethindene was a potent M2-selective antagonist (pA2 = 7.86/atria; pKi = 7.8/rat heart) with lower affinities for the M1 (pA2 = 6.36/rat duodenum; pKi = 7.1/NB-OK 1 cells), M3 (pA2 = 6.92/guinea-pig ileum; pKi = 6.7/rat pancreas) and M4 receptors (pKi = 7.0/rat striatum). It was more potent (up to 41-fold) than the (R)-isomer. In contrast, the stereoselectivity was inverse at ileal H1 receptors (pA2: (R)-isomer = 9.42; (S)-isomer = 7.48). Thus, (S)-dimethindene could be a valuable agent to test the hypothesis that M2 antagonists show beneficial effects in the treatment of cognitive disorders. It might also become the starting point for the development of diagnostic tools for quantifying M2 receptors in the CNS with PET imaging.     

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens.

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.     

Effects of aplysiatoxin and debromoaplysiatoxin on growth and differentiation of normal human bronchial epithelial cells

The effects of aplysiatoxin and debromoaplysiatoxin on the clonal growth rate, cross-linked envelope formation and plasminogen activator secretion of normal human bronchial epithelial cells were studied. Neither compound was mitogenic over a wide range of concentrations (10^–13 to 10^–7M). Both aplysiatoxin and debromoaplysiatoxin inhibited clonal growth rate with 50% inhibitory concentrations of 3 × 10^–11M and 10^–10M, respectively. Both compounds induced the formation of cross-linked envelopes and increased plasminogen activator secretion with equal potency. These data are similar to those previously obtained with 12-0-tetradecanoylphorbol-13-acetate and teleocidin B and suggest that aplysiatoxin and debromoaplysiatoxin induce terminal squamous differentiation in normal human bronchial epithelial cells.Abbreviations TPA 12-0-tetradecanoylphorbol-13-acetate - NHBE Normal Human bronchial epithelial - ID₅₀ 50% inhibitory concentration (dose) - PA Plasminogen activator - CLE Cross-linked envelope - LHC Laboratory of Human Carcinogenesis     

Distribution of cell surface lets protein in co-cultures of normal and transformed cells

When LETS protein positive and negative cells were co-cultured, the positive cells remained as positive and the negative cells remained as negative. Apparently the transformed cells do not secrete factors which are sufficient to influence the distribution of surface LETS protein on normal cells.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Size specific predation by herons and its effect on the sex-ratio of natural populations of the mosquito fish Gambusia affinis baird and girard

Summary Sex-ratios of Gambusia affinis populations in freshwater marshes in the Camargue (Rhône Delta), are highly biased in favour of males, whereas the sex-ratios in ditches are close to unity. Studies of the diet of free living birds and experimental studies on prey size selection in captivity show that the abnormal sex-ratios in marshes can be attributed to differential heron predation. Ditches are relatively free from predation. Mature female Gambusia are larger, and have an energy content 5–25 times greater than that of mature males. Handling times of Grey Heron (Ardea cinerea) and Little Egret (Egretta garzetta) are only slightly longer for female Gambusia than males. Hence, females represent a much more profitable prey.Analysis of nestling regurgitates show that Gambusia makes up a considerable proportion of the diet of four species of Camargue herons, and that the majority of Gambusia taken are females. Under experimental conditions, captive herons consume almost exclusively female fish, even when offered in ratios where they are heavily outnumbered by males.The relevance of these results to optimal diet theory is discussed.     

Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography.

Reaction of ceramides containing nonhydroxy fatty acids with benzoyl chloride in pyridine at 70 degrees C for 1 hr resulted in N-benzoylation to form N,N-acyl,benzoyl derivatives; O-benzoylation also occurred. However with ceramides containing 2-hydroxy fatty acids and phytosphingosine only O-benzoylation occurred even on prolonged treatment. Only O-benzoylation occurred on reaction with benzoic an hydride. However, the benzoylation of ceramides with phytosphingosine could not be achieved with benzoic anhydride and this benzoylation was performed by reaction with benzoyl chloride at 70 degrees C for 4 hr. Because N,N-acyl,benzoyl derivatives of ceramides containing nonhydroxy fatty acids produced by treatment with benzoyl chloride overlap methyl benzoate on high-performance liquid chromatography, benzoic anhydride was preferable for benzoylation of ceramides with nonhydroxy and 2-hydroxy fatty acids. On the other hand, the reaction with benzoyl chloride at 70 degrees C for 4 hr was used for quantitation of benzoylated ceramides containing 2-hydroxy fatty acids and phytosphingosine. 3-(p-Phenylbenzoyl)estrone was used as an internal standard for both reactions and values for ceramides containing 2-hydroxy fatty acids obtained by the two reactions were in good agreement. This procedure was applied to measurement of the ceramide levels in the brain, liver, and kidney of rats during development. The levels of ceramides containing nonhydroxy and 2-hydroxy fatty acids in the brain, liver, and kidney increased to the adult levels and then remained unchanged. Ceramide with phytosphingosine was detected in the liver and kidney, where its concentration gradually increased with age, but it was not found in the brain. The composition of nonhydroxy fatty acids were also analyzed.     

Strategies in bioreactor performance studies

As bioreactor performance is the result of the interactions between physiology and physical transport phenomena an adequate strategy for design and analysis of bioreactors has to be based on this interaction network. The paper summarizes this systematic approach based on a holistic view called “integrating strategy”. The strategy is outlined for the case of baker's yeast processing, where especially the comparability of kinetic models including the concept of “dynamic flux” is critically discussed showing that macro- and microkinetics have similar potential in describing bioprocesses according to the formal macroapproach.