Integrated membrane systems for gas separation in biotechnology: potential and prospects

Integrated non-porous membrane systems were applied for microbial combustible gas separation processes. Methane/CO₂ mixtures of various concentrations from methane fermentation processes (biogas) were separated using a membrane-separation complex of permabsorber type into individual components of technical grade (more than 95% purity). In experiments with three-component mixtures, using a selective membrane valve with various liquid carriers, all the gases of interest (H₂, CH₄ and CO₂) were obtained at greater than 90% purity in one separation step. The perspectives for the further application of non-porous membrane separating devices for various gaseous mixtures from different microbial processes are discussed.V. Teplyakov and E. Sostina are with the A.V. Topchiev institute of Petrochemical Synthesis, Russian Academy of Sciences, Membrane Research Center, Moscow 117912, Russia. E. Sostina is also, and A. Netrusov is with the Microbiology Department, Moscow University, Moscow 119899, Russia. I. Beckman is with the Chemistry Department, Moscow University, Moscow 119899, Russia.     

Qualitative mathematical discussion of different evolutionary states in water transport systems of plants

We shall present several qualitative mathematical models to describe the early evolution of water transport systems in plants. To perform this in a systematic way we apply methods which have been developed in phenomenological synergetics. These methods rest on the fact that it becomes possible to describe the macroscopic behavior of a complex system by a set of control and order parameters when they are suitably identified. Our presentation is addressed to community with interdisciplinary interests.     

The effect of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae

Summary The effects of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. Cells grown in the presence of acetic acid at a defined specific growth rate showed a higher acetic acid tolerance and a slightly lower trehalose content. Cells grown at a low specific growth rate showed a lower energy demand, a higher acetic acid tolerance, and a higher trehalose content. These results indicate that trehalose plays a growth rate dependent role in the tolerance of S. cerevisiae to acetic acid.     

Effects of UV-B radiation and nitrogen starvation on enzyme activities in isolated plasma membranes of Euglena gracilis

Circadian rhythms are characteristic of many physiological and biochemical processes in the freshwater flagellate Euglena gracilis. Earlier, we found that the rhythms of photosynthesis, phototaxis and cell shape followed the same pattern in control organisms, but were differently affected by stress such as UV-B irradiation and nitrogen deficiency. Here we extend our studies to use isolated plasma membranes to characterize the rhythms of some plasma membrane-bound enzymes. Also, we wanted to see whether stress-induced changes of these rhythms could be detected at the subcellular level and possibly be coupled to the changes seen in photosynthesis, phototaxis and cell shape. The isolation of plasma membranes using aqueous polymer two-phase partitioning was successful, as judged by the large enrichment of the plasma membrane-marker 5′-nucleotidase, and the difference in the polypeptide pattern compared with the microsomal fraction from which it was prepared. Two other enzymes were analyzed, K⁺, Mg²⁺-ATPase, and adenylyl cyclase. The specific activities of all three enzymes were decreased by UV-B radiation by ca 30–50%, compared with the control cultures. On the other hand, nitrogen deficiency not only reduced the activity of the K⁺.Mg²⁺-ATPase but also increased the activities of the 5′-nucleotidase and adenylyl cyclase. The different treatments also resulted in differences in polypeptide pattern, e.g., a polypeptide around 30 kDa seemed to be specific to plasma membranes of nitrogen-deficient cultures and one at 39 kDa for the UV-B radiated ones. All three enzymes showed diurnal rhythms that were affected by UV-B radiation. The peak in the rhythm of the ATPase was shifted by UV-B radiation, the rhythm of the 5′-nucleotidase nearly eliminated. The first peak of adenylyl cyclase activity was delayed, so that it looked more like a broad peak between 2 and 11 h after the onset of light. The rhythm of ATPase activity could be correlated with that of photosynthesis in both control and UV-B irradiated cultures. Also, the rhythms of adenylyl cyclase activity and cell shape changes showed some similarities.     

Peroxynitrite-Induced Cytotoxicity in PC12 Cells: Evidence for an Apoptotic Mechanism Differentially Modulated by Neurotrophic Factors

Abstract: Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 m M ) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC₅₀ = 850 µ M ) initially (3–4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.     

Catecholamine-synthesizing enzymes in the rat stomach

Local production of catecholamines in the stomach of the rat was studied by immunohistochemical demonstration of tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), the enzymes catalyzing the formation of dopamine, noradrenaline and adrenaline, respectively. A rich innervation of TH- and DBH-immunoreactive nerve fibers was seen in the muscular layers and the myenteric plexus, in the submucosa and in the walls of submucosal blood vessels and in the lamina propria at the base of the epithelial layer. In addition, TH-, but not DBH-immunoreactive nerve fiber networks surrounding ganglion cells in the myenteric plexus were frequently observed, indicating dopaminergic preganglionic innervation of the myenteric plexus. In the oxyntic epithelium, single TH- and DBH-immunoreactive fibers extended in the strands of lamina propria as far as the middle portion of the gastric glands. A small population of single angulate cells in the oxyntic epithelium showed TH-, but not DBH-immunoreactivity. No specific PNMT immunoreactivity was observed.     

Piccolyte Investments for Better Section Cutting

Piccolyte 115 (β-pinene polymers) added to Tissuemat, Paraplast or Peel-Away embedding media is recommended for investment of paraffin infiltrated tissues. Mixed with paraffin at 3% and 10% and used for double embedding of paraffin infiltrated tissues, Piccolyte 115 permits good, complete sections virtually free of folds or wrinkles in less time and with less effort than with paraffin embedding alone.     

Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.     

Protein extraction and activity in reverse micelles of a nonionic detergent

We describe, for the first time, the ability of a polyoxyethylene sorbitan trioleate-isopropanol microemulsion in hexane to solubilize pure proteins. The dependences of cytochrome c extraction and buffer solubilization by the reverse micellar system on ionic strength of the aqueous phase, detergent concentration, and cosurfactant concentration result in increased extraction. In addition, subtilisin (a serine protease) is shown to be active in this microemulsion. Further the activity of the enzyme can be regulated by the water content of the micelles, enabling control of enzyme activity by "solvent engineering."     

Biocatalytic synthesis of acrylates in organic solvents and supercritical fluids: I. optimization of enzyme environment

Biocatalytic transesterification of methylmethacrylate is possible in many different solvents. The reaction rate is readily controlled by variation in solvent physical properties. The reaction proceeds better in hydrophobic solvents, and activity can be restored in hydrophilic solvents by the addition of water. We have now demonstrated that supercritical carbon dioxide is not a good solvent for the reaction between 2-ethlhexanol and methylmethacrylate. It apperars that the supercritical carbon dioxide may either alter the pH of the microaqueous environment associated with the protein or reversibly form covalent complexes with free amine groups on the surface of the enzyme. Although supercritical carbon dioxide is a poor solvent for acrylate transesterification, many other supercritical fluids (ethane, ethylene, sulfur hexafluoride, and fluoroform) are better than most conventional solvents. In supercritical ethane it is possible to control the activity of the enzyme by changing pressure, and the enzyme appears to follow Michaelis-Menten Kinetics. We find that sulfur hexafluoride, the first anhydrous inorganic solvent in which biocatalytic activity has been reported, is a better solvent than any conventional or supercritical organic fluid tested.