Extension of cell membrane boosting squalene production in the engineered Escherichia coli

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.     

Pattern of tonic activity in different groups of rabbit superior cervical ganglion neurons

Tonic activity in rabbit superior cervical ganglion neurons was investigated using intracellular recording techniques as well as changes produced when the animal breathed a gaseous mix with a raised CO₂ level. The test neurons were divided into three groups depending on the pattern of their tonic activity and reflex change. Action potentials were produced by the activity of dominant and accessory preganglionic inputs in the firing pattern of all neuronal groups, implying the existence of other types of inputs into the neurons innervating different organs. Having analyzed changes in action potential rate and EPSP in the tonic activity of neurons from different groups, it was presumed that preganglionic fibers with a similar activity pattern converge on the majority of neurons in each group.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 5, pp. 665–672, September–October, 1987.     

Blocking action of Nephila clavata spider toxin on ionic currents activated by glutamate and its agonists in isolated hippocampal neurons

The blocking action ofNephila clavata spider neurotoxin, or JSTX, on ionic currents activated by L-glutamate and its agonists when applied to the membrane of neurons isolated from the rat hippocampus was investigated using a concentration clamp technique. Crude JSTX venom was found to block L-glutamate-, quisqualate, and kainate-activated ionic currents induced by activating non-N-methyl-D-aspartate (non-NMDA) membrane receptors. Following the effects of JSTX, ionic currents activated by L-glutamate and its agonists declined to 34–36% of their initial value with no recovery during JSTX washout. An active fraction of JSTX at concentrations of 10^–4–10^–5 produced almost total but partially reversible blockade of ionic currents. The action of JSTX became less effective during depolarization. The concentration dependence of JSTX-induced blockade of kainate-activated ionic currents was investigated and the velocity constants of interaction between the toxin and glutamate receptors obtained. It is postulated that JSTX interacts with chemically-operated non-NMDA ionic channels, blocking their transition into a number of their possible open states.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 152–160, March–April, 1989.     

Argiopine,argiopinines, and pseudoargiopinines as glutamate receptor blockers in hippocampal neurons

A homologous set of low-molecular weight compounds selectively blocking ionic currents were purified from venom from the spiderArgiope lobata with a selective blocking action on ionic currents activated by applying glutamate and its agonist kainic acid (KA) to the membrane of neurons isolated from the rat hippocampus. Three groups of these compounds — argiopine, argiopinines, and pseudoargiopinines, produced voltage-dependent glutamate- and KA-activated ionic currents at concentrations of 10^–6-10^–4 M, interacting primarily with agonist-activated ionic channels without affecting K_d values of the agonist. The blocking action could be partially reversed by argiopine application but only slightly when argiopinines and pseudoargiopinines were used. Kinetics of toxin effects on Ka-activated ionic currents showed at least two exponential components with different time constants. Simple and reversed rate constants of interaction between toxins and ionic channels were estimated from the plot of the kinetics of ionic current blockade and recovery against toxin concentration. Argiopine, argiopinines, and pseudoargiopinines lend themselves to further research into glutamate receptors of the mammalian CNS employing electrophysiological and biochemical techniques.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev, M. M. Shemyakin Institute of Bioorganic Chemistry, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 748–756, November–December, 1989.     

Heterochromatic regions in different Drosophila melanogaster stocks contain similar arrangements of moderate repeats with inserted copia-like elements (MDG1)

Seven out of twenty 30–50 kb genome fragments with an MDG1 copia-like element cloned in cosmids were found to carry homologous sequences which belong to a new family of non-mobile heterochromatic moderate repeats (the HMR family). These repeats along with the MDG1 copies inserted in them are under-replicated in polytene chromosomes. Such repeats may also be located in the intercalary heterochromatin site 12E of the X chromosome. Chromosomal heterochromatic regions are enriched with one of the two main genomic variants of MDG1, MDG1^het, identifiable by EcoRI restriction. From Southern DNA blot analysis the number of MDG1^het copies and their sites within the heterochromatin are invariant in all the stocks examined, while there is not a single MDG1 site along the polytene chromosomes shared by all the stocks in question.     

Hot spot of recombination within DXS164 in the Duchenne muscular dystrophy gene.

The DMD gene, which spans more than 2,000 kbp, has been assigned to band Xp21 of the X chromosome. Two subclones (PERT 87-1 and PERT 87-15) of the intragenic locus DXS164 physically are separated by approximately 60 kbp. Linkage studies were done in 49 informative DMD families by using the LINKAGE program. Crossing-over between the loci studied occurred in four families. A recombination rate of 4% (support interval [Zmax-1] 1%-10%), which was 54 (support interval 14-135-fold) times higher than expected, was found with a maximum lod score of 13.50. These data suggest a hot spot for recombination within DXS164.     

Excitation of feline cutaneous mechanosensitive C-fiber sensory units by intraarterial administration of potassium ions

Response of low- and high-threshold mechanosensitive C fiber sensory units (MSU) in n. saphenus to close-arterial injection of potassium ions in subnoxious and noxious concentrations (SC and NC) were investigated in anesthetized cats. Two groups of high-threshold units were found: 1) MSU reacting to K⁺ at both SC and NC, but responding very differently to each of these concentrations and 2) MSU responding to NC only. Findings would indicate that high-rather than lowthreshold MSU participate in the perception of noxious stimuli. The possibility of differentiation between subnoxious and noxious stimuli by a proportion of high-threshold MSU is considered. High-threshold MSU discharges accompanying excitation of these units by subnoxious and noxious chemical stimuli are quantified.National Cardiology Research Center, Academy of Medical Sciences of the USSR, Moscow. translated from Neirofiziologiya, Vol. 20, No. 2, pp. 147–154, March–April, 1988.     

Biochemical Mapping of Peptidyl-Glycine α-Amidation Activity in the Rat CNS

Peptidyl-glycine alpha-amidation enzyme activity has been measured in 36 nuclei or areas in the rat CNS and pituitary using D-Tyr-Phe-Gly as the substrate. The distribution of this enzyme is highly uneven, with highest activity levels (greater than 30 pmol/mg of protein/h) in hypothalamic nuclei, substantia grisea centralis, and nucleus ruber; moderate activity levels (10-30 pmol/mg of protein/h) in globus pallidus, septum, midbrain, pons, medulla oblongata, and cervical spinal cord; and low activity levels (1-10 pmol/mg of protein/h) in other telencephalic and thalamic structures. Almost no alpha-amidation activity (less than 0.5 pmol/mg of protein/h) was detected in cerebellar cortex. The Km values in several brain regions are of the same order.