A model for a non-chemical form of life: Crystalline physiology

A definition of fundamental living units is given according to which they are constituted by the material support of some ‘memory’ the latter is required

- to be stable,

- to contain rich information,

- to diffuse it into the surrounding medium.

It is then shown that the complex dislocation networks encountered in crystals can in some cases follow these criteria and lead to a crystalline physiology. The places of possible occurrence in nature of this kind of physiology, terrestrial and extraterrestrial rocks, interplanetary dust, white dwarfs and neutron stars are then discussed.     

The Acute and Chronic Effects of Monosodium l-Glutamate on Serum Iron and Total Iron-Binding Capacity in the Jugular Artery and Vein of Pigs

We analyzed the effects of acute and chronic oral administration of monosodium l-glutamate (MSG) on serum iron (Fe) levels and total iron-binding capacity (TIBC) in piglets. In the first experiment, 12 piglets were randomly assigned to two groups: one fed a standard diet (SD) and the other fed an SD containing MSG (10 g/kg). On day 30, serum, liver, kidney, and spleen samples were collected to determine the Fe levels. In the second experiment, six pigs were surgically fitted with a catheter in the jugular artery and vein to investigate the dynamic changes of serum Fe and TIBC. Blood samples were taken from each pig via the catheter every 30 min, for a period of 4 h. The results show that MSG increases Fe levels in the spleen (P?<?0.05) and in serum obtained from the jugular artery (P?<?0.01). In addition, TIBC in serum obtained from the jugular artery demonstrated an increasing trend in pigs fed the MSG diet; however, this trend was not observed in the jugular vein. In conclusion, MSG increases Fe retention by enhancing TIBC in serum.     

Regulation of l-Glutamate Biosynthesis by Copper Ions

A specific regulatory effect of copper ions on the microbiological synthesis of l-glutamate from acetate was found. The minimal concentration of copper ions necessary for the maximal production of l-glutamate was about 0.025 µg/ml at which the yield of l-glutamate was four times greater than that in the absence of copper ions. This effect of copper was demonstrated only when acetate was the substrate; it was not observed when the substrate was glucose ethanol, lactate or n-paraffin.

The physiological features of the l-glutamate production from acetate were examined in the presence or absence of copper ions. The most striking features of the culture without added copper ions were the increase in Q_O2 and NADH oxidase and the marked reduction of succinate oxidase accompanied with the reduction of l-glutamate formation. In addition, the regulation of l-glutamate synthesis by copper ions proved to have no relation to the wellknown regulatory factor, cell permeability. These facts suggest that the l-glutamate biosynthesis from acetate is regulated through unknown factors related to the respiratory activities.     

A Plastidial Localization and Origin of l-Glutamate Dehydrogenase in a Soybean Cell Culture

The subcellular distribution of l-glutamate dehydrogenase (GDH, EC 1.4.1.3.) was studied in SB3 soybean (Glycine max) cells using subcellular fractionation techniques. Compounds that inhibit protein synthesis either on 80s or 70s ribosomes were also used to give a preliminary idea of which subcellular fraction is involved in GDH synthesis. It was found that whereas cycloheximide and puromycin considerably reduced the total amount of protein synthesized by the cells, they did not appear to inhibit the synthesis of GDH. In the presence of chloramphenicol, both GDH activity and protein level in the cells were considerably reduced, suggesting that this enzyme was synthesized in organelles and not in the cytosol. Streptomycin, which inhibits plastid protein synthesis, also inhibited synthesis of GDH, indicating that a fraction of GDH activity was plastidial in origin. This is supported by the data on subcellular distribution of the enzyme, which showed that a major fraction of GDH is found in the plastidial fraction, although some activity is found associated with the mitochondrial fraction also. Since a major fraction of GDH activity was found in the plastidial fraction, we studied protein synthesis using isolated plastids and ³⁵S-methionine. Using antibodies raised against purified GDH, we identified a ³⁵S-labeled 41-kilodalton polypeptide synthesized by plastids as GDH.     

Crystalline alkaline form fructose-1,6-diphosphatase. A simple purification procedure and preliminary x-ray diffraction analysis.

A simple and rapid procedure has been found for the preparation of higly pure alkaline form fructose-1,6-diphosphatase from turkey liver. The protein is tetrameric and is composed of subunits comprised of a 29,000 molecular weight core fragment and an associated 7,000-dalton S-peptide. Large single crystals of the protein, suitable for x-ray diffraction analysis, are produced in the course of the preparation. These are of space group R3 and have rhombohedral cell dimensions of a = 153 A and delta = 163 degrees (equivalent hexagonal dimensions a = 303 A, c = 75 A). There is one entire tetramer of 144,000 daltons in the asymmetric unit. The enzymatic properties of this enzyme appear to be very similar to those reported for the protein from other organisms, but its unique propensity to crystallize opens the way for a full three-dimensional structural analysis.     

Crystalline morphology of trihexosylceramide deposits characteristic of Fabry's disease (notably the rheumatic form)

This paper deals with the various crystalline aspects of the ceramide trihexoside (CTH) inclusions, in the synovial tissue essentially. A basic classification is proposed and uncommon examples are illustrated. Image processing confirms the membrane-like structure of CTH inclusions and corroborates the proposed classification.     

Intracellular Localization of Three l-Glutamate Dehydrogenase Isozymes from Chlamydomonas reinhardtii

The intracellular localization of the activity and synthesis of three isozymes of NAD(P)⁺-glutamate dehydrogenase from the unicellular green alga Chlamydomonas reinhardtii cw-92 has been established. Isozyme activities have been located within mitochondria by using differential centrifugation techniques and discontinuous Percoll gradient separations. Experiments with protein synthesis inhibitors cycloheximide, rifampicin, chloramphenicol, and actinomycin D, under dark and carbon starvation conditions, revealed that synthesis of the three isozymes was likely to occur in cytosol as precursor proteins that are then transported and processed inside the mitochondria.     

A Crystalline P-Protein in Gmelina arborea

In a light microscope study of the secondary phloem in Gmelinaarborea (Verbenaceae) many sieve elements were found to possessbar-shaped cytoplasmic inclusions of proteinaceous nature Itis suggested that these inclusions represent a type of crystallineP-protein not reported in the family Verbenaceae before Crystalline P-protein, phloem, sieve element inclusion     

A Bioluminescence Method for the Measurement of l-Glutamate: Applications to the Study of Changes in the Release of l-Glutamate from Lateral Geniculate Nucleus and Superior Colliculus After Visual Cortex Ablation in Rats

We have developed a rapid, simple, specific, and very sensitive bioluminescence method for the measurement of L-glutamate (L-Glu). Oxidation of L-Glu by glutamate dehydrogenase has been coupled with bacterial FMN reductase and luciferase. Light production (i.e., peak height or integral) was linear from less than 0.5 to 500 pmol of L-Glu. Potential interfering substances that may be encountered in brain tissue have been identified. The most potent inhibitors were ascorbate and the biogenic amines. Procedures that conferred long-term stability of the reagent mixture (greater than 8 h) were established. Bioluminescence analysis of L-Glu content in brain tissue extracts, fractions from release experiments, and human CSF corroborated respective results obtained by HPLC analysis. In this study, we have applied the method to monitor changes in the KCl-evoked release of endogenous L-Glu from milligram amounts of brain tissue, i.e., from lateral geniculate nucleus and superior colliculus after visual cortex ablation.     

l-Glutamate Uptake Inhibitors May Stimulate Phosphoinositide Hydrolysis in Baby Hamster Kidney Cells Expressing mGluR1a via Heteroexchange with l-Glutamate Without Direct Activation of mGluR1a

Abstract: The functional efficacies of inhibitors of l -glutamate uptake for altering second messenger formation in baby hamster kidney cells expressing subtypes mGluR1a, mGluR2, and mGluR4 of the metabotropic glutamate receptor family were examined. l -Serine-O-sulfate was an agonist at mGluR1a (EC₅₀ = 70 µM), mGluR2 (EC₅₀ = 25 µM), and mGluR4 (EC₅₀ = 324 µM). l -Cysteine sulfinate, 1-aminocyclobutane-trans-1,3-dicarboxylate, l -cysteine, and dl -threo-3-methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC₅₀ values of 43, 64, 463, and 488 µM, respectively, and displaced l -[³H]glutamate binding from membranes prepared from these cells with respective IC₅₀ values of 48, 44, 79, and 139 µM. However, d -aspartate,l -trans-pyrrolidine-2,4-dicarboxylate, l -threo-3-hydroxyaspartate, and l -aspartate-β-hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC₅₀ values of 73, 54, 57, and 430 µM) but did not displace l -[³H]glutamate binding. These compounds inhibited Na⁺-dependent l -glutamate uptake into baby hamster kidney cells with IC₅₀ values similar to those for stimulation of phosphoinositide hydrolysis in mGluR1a cells. Phosphoinositide hydrolysis in mGluR1a cells, as stimulated by inhibitors of (or substrates for) this l -glutamate transporter, was significantly attenuated in the presence of l -glutamate decarboxylase (EC 4.1.1.15) or l -alanine aminotransferase (EC 2.6.1.2). Furthermore, incubation with 1 mMl -trans-pyrrolidine-2,4-dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 ± 0.2 µM) in the assay buffer four- to fivefold as measured by HPLC analysis. Thus, heteroexchange with endogenous l -glutamate may lead to erroneous estimations of the functional efficacies at mGluR1a.     

New crystal form of recombinant murine interferon-beta

Although we have reported (Matsuda, S., Kawano, G., Itoh, S., Mitsui, Y., and Iitaka, Y. (1986) J. Biol. Chem. 261, 16207-16209) that recombinant murine interferon-beta produced in Escherichia coli was crystallized in an orthorhombic space group C222(1) using polyethyleneglycol 8000 as precipitant, the crystals had an insufficient resolution and a marked tendency for orientational disorder around the c axis. We now report that another form of murine interferon-beta crystals with little disorder was obtained in the presence of dioxane using ammonium sulfate as precipitant. The new crystals belong to a hexagonal space group P6(1) or P6(5) with a = b = 71.4 A and c = 79.6 A having only one murine interferon-beta molecule in an asymmetric unit. The crystals are reasonably stable to x-rays and significantly diffract up to 2.2 A resolution when a synchrotron beam is used.     

Fermentative Production of l-Glutamine by Sulfaguanidine Resistant Mutants Derived from l-Glutamate Producing Bacteria

Excellent l-glutamine producers were screened for among sulfaguanidine resistant mutants derived from the wild type l-glutamic acid-producing bacteria, Brevibacterium flavum, Brevibacterium lac to fermentum, Corynebacterium glutamicum and Microbacterium ammoniaphilum.

The best strain, No. 1~60, was a sulfaguanidine resistant mutant derived from B. flavum 2247 by mutation. Strain No. 1~60 accumulated 41.0 mg/ml of l-glutamine after 48 hr of cultivation from 10% glucose as a carbon source. This yield was the highest among those so far reported.

The addition of Mn^{2 +} (2 ppm) to the standard medium for B. flavum 2247 decreased the l- glutamine production and increased the l-glutamic acid excretion markedly. On the contrary, strain 1 —60 was not affected the Mn²⁺ (2 ppm) addition at all.

Glutamate kinase activity and the intracellular content of ATP in sulfaguanidine resistant mutant No. 1~60 were higher than those in the parent strain, B. flavum 2247.

It was confirmed that the increase in glutamate kinase and the increase in internal ATP, which were important for the l-glutamine synthesis, were very effective for the improvement of l-glutamine-producing mutants.     

Synthesis and Reaction Chemistry of Nanosize Monosodium Titanate

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the material with Au(III) ions. The synthesis method was derived from a sol-gel process used to produce micron-sized monosodium titanate (MST), with several key modifications, including altering reagent concentrations, omitting a particle seed step, and introducing a non-ionic surfactant to facilitate control of particle formation and growth. The resultant nMST material exhibits spherical-shaped particle morphology with a monodisperse distribution of particle diameters in the range from 100 to 150 nm. The nMST material was found to have a Brunauer-Emmett-Teller (BET) surface area of 285 m²g^-1, which is more than an order of magnitude higher than the micron-sized MST. The isoelectric point of the nMST measured 3.34 pH units, which is a pH unit lower than that measured for the micron-size MST. The nMST material was found to serve as an effective ion exchanger under weakly acidic conditions for the preparation of an Au(III)-exchange nanotitanate. In addition, the formation of the corresponding peroxotitanate was demonstrated by reaction of the nMST with hydrogen peroxide.