Preparation of Porcine Neurophysin Proteins by High Performance Liquid Chromatography

Abstract: The crude neurophysin containing extract from posterior lobes of porcine pituitaries was roughly purified by gel chromatography. 15 mg of the lyophilized neurophysin complex were completely separated by HPLC yielding in neurophysin I₁ (3.6 mg), I₂ (4.0 mg), II (4.6 mg) and III (1.9 mg). All of the neurophysins were homogenous by PAGE and SDS-electrophoresis, isoelectrofocussing, amino-acid composition and N- and C-terminal amino acid analysis. In conclusion, HPLC is a reliable and quick method for the preparation of pure neurophysins.     

Cherry fruit development: Indoleacetic acid oxidase isoenzymes in the seed

Two anionic indoleacetic acid oxidase isoenzymes were separated by polyacrylamide gel electrophoresis from an acetate buffer (0.2 M, pH 4.0) extract of sour cherry ( Prunus cerasus L. cv. Montmorency) seed. One isoenzyme migrated to Rf 0.25 (I1) and the other to Rf 0.78 (I₂). Isoenzyme I, exhibited hyperbolic kinetics and was found during all three stages of fruit development with the highest levels during early stage II. The isoenzyme I₂ showed sigmoidal kinetics and was found only during stages II and III of fruit growth with highest levels during stage III. The activities of both isoenzymes were markedly enhanced by addition of Mn2+ and 2,4-dichloro-phenol to the reaction mixture. Isoenzyme I, showed higher affinity for indoleacetic acid than isoenzyme I₂. The significance of these isoenzymes in cherry fruit growth is discussed.     

Low temperature spectrophotometry of the phototransformation of Pfr to Pr in pelletable pea phytochrome

Manabe, K. 1987. Low temperature spectrophotometry of the phototransformation of P_fr to P_r, in pelletable pea phytochrome.
Low temperature spectrophotometry was used to study the phototransformation of P_fr to P_r in 1000–7000 g pelletable fractions extracted from dark grown pea ( Pisum sativum L. cv. Alaska) epicotyls which had been irradiated with red and then far-red light. At -170°C, far-red irradiation of the pelletable phytochrome which had been pre-irradiated with saturating fluence of red light before freezing caused formation of an intermediate (named I₆₆₀), the difference spectrum of which showed a marked ab-sorbance decrease at 740 nm and a concomitant small increase at about 660 nm. The inermediate I₆₆₀ was converted to another intermediate (I₆₆₀) when it was warmed above -80°C. The difference spectrum of this intermediate showed a positive peak at 670 nm. This intermediate was photoconverted to P_fr by red irradiation and also underwent dark reversion to P_fr at -60°C. I₆₆₀ formed P_r if the temperature was above -10°C. The basic features of the phytochrome intermediates resemble those obtained in vivo and in degraded purified phytochrome.     

Characterization of extracellular oxygen consumption by the green alga Selenastrum minutum

Cells of the green alga Selenastrum minutum display a high capacity for extra-mitochondrial O₂ consumption in the presence of effectors such as salicylhydroxamic acid and/or NADH. We provide evidence that this O₂ consumption is mediated by extracellular peroxidase. Peroxidase capacity, measured as the potential for stimulation of O₂ consumption by a combination of salicylhydroxamic acid and NADH, changed over a 10-day time course. Maximal stimulation of O₂ consumption occurred at day three, at which point the capacity for peroxidase-mediated O₂ consumption was three-to four-fold higher than that of the control O₂ consumption rate. Peroxidase-mediated O₂ consumption was sensitive to inhibition by 50 m M ascorbate and by cyanide. Cyanide titration curves indicated that O₂ consumption by peroxidase was much more sensitive to inhibition by cyanide than was O₂ consumption by cytochrome oxidase (I₅₀ < 1.6 μ M and I₅₀= 18.3 μ M cyanide, respectively). By using evidence from a combination of cyanide titration curves and ascorbate inhibition, we concluded that despite a large capacity for peroxidase-mediated O₂ consumption, peroxidase did not measurably contribute to control rates of O₂ consumption. In the absence of effectors, O₂ consumption was mediated primarily by cytochrome oxidase.     

Effects of mitochondrial complex III disruption in the respiratory chain of Neurospora crassa

In mitochondria from most organisms, including Neurospora crassa , dimeric complex III was found associated with complex I. Additional association of complex IV with this core structure leads to the formation of a respirasome. It was recently described for bacteria and mammals that complex III is needed for the assembly/stability of complex I. To elucidate the role of complex III in the organization of the respiratory chain of N. crassa , we analysed strains devoid of either the Rieske iron-sulphur or the COREII polypeptide subunits. The mutants display reduced growth, are female sterile and lack active complex III. The supramolecular organization of the oxidative phosphorylation system was characterized by electrophoretic analyses and the efficiency of the respiratory chain analysed by oxygen consumption measurements. The results obtained indicate that absence of complex III activity is not associated with the absence of complex I or complex IV, and leads to the induction of alternative oxidase. Complex III mutant mitochondria are devoid of respirasomes but contain significant amounts of dimeric complex I (I₂) and of the supercomplex I₁IV₁. Moreso, for the first time the alternative oxidase was found associated with dimeric complex IV and with supercomplex I₁IV₁.     

Cercospora beticola toxins. IX. Relationship between structure of beticolins, inhibition of plasma membrane H+-ATPase and partition in lipid membranes

Beticolins are yellow toxins produced by the fungus Cercospora beticola . The effect of one of them, beticolin-1. has been investigated on corn root plasma membrane H⁺-ATPase (EC 3.6.1.35) at different purification levels (plasma membrane fraction, partially, or highly purified enzyme). The results obtained demonstrated that (1) the purified proton pump was inhibited directly by low amounts of the toxin (I₅₀= 1.62 ± 0.18 μM), (2) the biological effects of beticolin-1 were similar to those of CBT ( Cercospora beticola toxin). Furthermore, it was established that the efficiency of the different beticolins was clearly related to their ability to interact with the lipid bilayers, determined by fluorometric studies: the toxins that exhibited the lower I₅₀ (50% inhibitory concentrations) values were the molecules that had the lowest partition coefficient to liposomes.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D

Abstract Poly(3-hydroxybutyric acid) granules, which harbored only four major granule-associated proteins as revealed by SDS polyacrylamide gel electrophoresis, were isolated from crude cellular extracts of Chromatium vinosum D by centrifugation in a linear sucrose gradient. N-Terminal amino acid sequence determination identified two proteins of M _r 41 000 and M _{r 40 000} as the phaE _Cv and phaC _Cv translational products, respectively, of C. vinosum D. In a previous study it was shown that both proteins are required for the expression opf poly(3-hydroxyalkanoic acid) synthase activity. The N-terminus of the third protein ( M _r 17 000) exhibited no homology to other proteins. Lysozyme, which was during purification of the granules, exhibited a strong affinity to PHB granules and was identified as the fourth protein enriched with the granules.     

Trifluoperazine-Sensitive Step during Sea Urchin, Echiuroid and Pelecypod Egg Activation

Trifluoperazine, a calmodulin-antagonist, is shown to inhibit egg activation by ionophore A 23187 in sea urchin (I₅₀: 43 μM), by trypsin in echiufoids (I₅₀: 22 μM) and by KCl in bivalves (I₅₀: 34 μM). In each case the inhibition could be reversed by washing the eggs and the trifluoperazine-sensitive period was clearly limited. In Barnea and Urechis , trifluoperazine inhibits calcium uptake. A common trifluoperazine-sensitive step, possibly involving calmodulin, may thus be shared by a variety of animal groups during egg activation.     

Alterations in reactivity of the blood factors of cattle red cells after pronase treatment

Pronase treatment of cattle red cells produced various effects: (a) an increase in reactivity of the J factor and evolution of a specific cryptoantigen; (b) decrease in the A-B, G₂, K, I₂ O₂, O₃, P, Q, T_1; Y₂, A, B', E'₂, E'₃, I', K', O'-L'-V-L and M, factors, but (c) no change in the specifity or in the titre of the remaining 16 different blood factors. Most of the pronase-affectable blood factors were destroyed in a rather narrow but characteristic range of pronase treatment intensities. However, at like intensities, variations were demonstrable due to the fact that the blood factor occurred (a) in red cells from different individuals, and (b) in different phenogroups or subgroups of the B locus.     

Intracellular Microinjection of Anticalmodulin Drugs Does not Inhibit the Cortical Reaction Induced by Fertilization, Ionophore A 23187 or Injection of Calcium Buffers in Sea Urchin Eggs

The drugs, fluphenazine, chlorpromazine, dibucaine, propranolol, vinblastine and W7[N-(6-arninohexyl)-5 chloro-1-napthalene-sulfonamide], which have been shown to prevent formation of the ternary activated complex of Ca⁺⁺-calmodulin with several soluble or membrane proteins, inhibit the cortical reaction induced by fertilization, by ionophore A 23187 or by the microinjection of Ca⁺⁺ buffers when applied from outside to sea urchin eggs. In contrast, direct intracellular microinjection of these drugs, even at concentrations much exceeding their I₅₀ for external application, does not suppress elevation of the fertilization membrane, although it prevents cleavage after fertilization. The implication is that intracellular calmodulin is not the receptor of Ca⁺⁺ in the Ca⁺⁺-dependent exocytosis of cortical granules induced by fertilization, by ionophore, or by the micro-injection of calcium buffers.     

Physiological Effects on Periplaneta americana of Bacillus sp. Exotoxin

ABSTRACT This study was performed to evaluate the physiological effects of Bacillus sp. on Periplaneta americana. The insecticidal exotoxin was fractionated by ion-exchange chromatography and gel filtration from Bacillus sp. cultivates. Lp₂ among 4 lipoproteins showed markable increase after 24 hrs, but there was no change in glycoprotein. The enzyme activities of P. americana showed mostly decreasing pattern by treatment of exotoxin fractions. Protease activities decreased at 12 hrs after treatment in comparison with normal subjects. Amylase also showed decreasing pattern and maximal decline was observed after 12 hrs. Trehalase activities decreased at 6 hrs and 24 hrs. Invertase activities decreased from 6 hrs to 24 hrs, but recovered after 48 hrs. Amylase isozyme A₁ and A₂ disappeared and new isozyme appeared after 12 hrs with exotoxin treatment. Trehalase isozyme T₁ and invertase I₄ increased markedly after 12hrs. However, non-specific esterase E1 disappeared after 24hrs.     

Prostanoid production in Saccharomyces cerevisiae provides a novel assay for nonsteroidal anti-inflammatory drugs

Prostanoids are a large family of lipid mediators originating from prostaglandin H synthase (PGHS) activity on the 20-carbon polyunsaturated fatty acids dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid. The two mouse PGHS isoforms, PGHS-1 and PGHS-2, were expressed in Saccharomyces cerevisiae (yeast), as was a signal-peptide-deleted version of PGHS-1 (PGHS-1MA). PGHS-1 showed high activity with both AA and DGLA as substrate, whereas PGHS-2 activity was high with DGLA but low with AA. Signal peptide removal reduced the activity of PGHS-1MA by >50% relative to PGHS-1, but the residual activity indicated that correct targeting to the lumen of the endoplasmic reticulum may not be necessary for enzyme function. Coexpression of PGHS-1 with cDNAs encoding mouse prostaglandin I synthase and thromboxane A synthase, and with Trypanosoma brucei genomic DNA encoding prostaglandin F synthase in AA-supplemented yeast cultures resulted in production of the corresponding prostanoids, prostaglandin I₂, thromboxane A₂ and prostaglandin F_2α. The inhibitory effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on prostanoid production were tested on yeast cells expressing PGHS-1 in AA-supplemented culture. Dose-dependent inhibition of prostaglandin H₂ production by aspirin, ibuprofen and indomethacin demonstrated the potential utility of this simple expression system in screening for novel NSAIDs.     

Triiodothyronine Is a High-Affinity Inhibitor of Amino Acid Transport System L1 in Cultured Astrocytes

Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T₃) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na⁺-independent, and efficiently inhibited by T₃ (half-inhibition at ∼ 2 μ M ). Two Na⁺-independent L-like systems (L₁ and L₂), common to leucine and aromatic amino acids, were characterized kinetically. System L₂ had a low affinity for leucine and tryptophan ( K _m= 0.3–0.9 m M ). The high-affinity system L₁ ( K _m∼ 10 μ M for both amino acids) was competitively inhibited by T₃ with a K _i of 2–3 μ M (close to the T₃ transport K _m). Several T₃ analogues inhibited system L₁ and the T₃ transport system similarly. Glutamine uptake and α-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T₃ had little effect on the uptakes of glutamine and α-(methylamino)isobutyric acid. The results indicate that the T₃ transport system and system L₁ are related.     

Chromatographic Discrimination of Soluble Neuropathy Target Esterase Isoenzymes and Related Phenyl Valerate Esterases from Chicken Brain, Spinal Cord, and Sciatic Nerve

Abstract: Neuropathy target esterase (NTE) activity is operatively defined in this work as the phenyl valerate esterase (PVase) activity resistant to 40 µ M paraoxon but sensitive to 250 µ M mipafox. Gel filtration chromatography with Sephacryl S-300 of the soluble fraction from spinal cord showed two PVase peaks containing NTE activity (S-NTE1 and S-NTE2). The titration curve corresponding to inhibition by mipafox was studied over the 1–250 µ M range, in the presence of 40 µ M paraoxon. The data revealed that S-NTE1 and S-NTE2 have different sensitivities to mipafox with I₅₀ (30 min) values of 1.7 and 19 µ M , respectively. This was similar to the pattern observed in the soluble fraction from sciatic nerve with two components ( V _o peak, or S-NTE1; and 100-K peak, or S-NTE2) with different sensitivity to mipafox. However, in the brain soluble fraction, only the high-molecular-mass (>700-kDa) peak or S-NTE1 was obtained. It showed an I₅₀ of 5.2 µ M in the mipafox inhibition curve. The chromatographic profile was different on changing the pH in the subcellular fractionation. When the homogenized tissue was centrifuged at pH 6.8, the V _o peak activity decreased in the soluble fraction from these nerve tissues. This suggests that the V _o peak could be related to materials partly solubilized from membranes at higher pH. The chromatographic pattern and mipafox sensitivity suggest that the different tissues have a different NTE isoform composition. S-NTE2 should be a different entity than S-NTE1 and particulate NTE. The potential role of soluble forms in the mechanism of initiation or promotion of neuropathy due to organophosphorus remain unknown.     

Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.     

Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4

A protease-sensitive antibacterial substance produced by Bacillus coagulans I₄ strain, isolated from cattle faeces, was classified as a bacteriocin-like inhibitory substance and named coagulin. The inhibitory spectrum included B. coagulans and unrelated bacteria such as Enterococcus , Leuconostoc , Oenococcus , Listeria and Pediococcus . Coagulin was stable at 60 °C for 90 min, at a pH ranging from 4 to 8 and appeared to be unaffected by α-amylase, lipase or organic solvents (10% v/v). Coagulin exhibited a bactericidal and a bacteriolytic mode of action against indicator cells. The apparent molecular mass was estimated to be about 3–4 kDa by SDS-PAGE. The B. coagulans I₄ strain harbours a plasmid, pI₄, approximately 14 kb in size. Novobiocin curing experiments yielded two derivatives that no longer produced the bacteriocin-like inhibitory substance. Plasmid content of these two derivatives showed that one had lost pI₄,whereas the second harboured a deleted form of this plasmid, thus suggesting a plasmid location for the genes for coagulin production.     

Structure and Macromolecular Composition of the Jelly Coats of the Urodele Ambystoma mexicanum

Cleavage-stage embryos of the neotenic urodele Ambystoma mexicanum are surrounded by a fertilization envelope and four macroscopic jelly coats termed J₁ (innermost) through J₄ (outermost). In sections prepared for light microscopy, each of the jelly layers stained with protein stains and the periodic acid-Schiff's reagent, but only J₁ stained with alcian blue at pH 2.5. These results suggest that each layer consists of proteins and glycoproteins and that J₁ uniquely contains some sulfate esters. Only J₄ was solubilized with alkaline mercaptan treatment in situ , however, the isolated inner jelly complex (J₁, J₂ and J₃) was easily dissolved in this reagent suggesting that solvent access is impaired in situ . A single alcian blue-staining component plus one protein-staining component were detected on reducing polyacrylamide gel electrophoresis of outer jelly (J₄). In the inner jelly complex (J₁, J₂, J₃), two protein-staining components were detected and no alcian blue-staining components were observed. A predominant polypeptide of 110,000 molecular weight was detected and purified to homogeneity on reducing and denaturing gels of the inner jelly complex. Amino acid analysis of the polypeptide demonstrated a slightly higher fraction of acidic over basic amino acids (Glx+Asx=18.1 mole% vs . Arg + Lys = 11.7 mole%). The N-terminal amino acid was Glu and the sequence of the first eleven amino acids was determined.     

Influence of nitrate and ammonium on the growth and 2,4-diaminobutyric acid composition of flatpea (Lathyrus sylvestris L.)

Abstract. Presence of 2.4-diaminobutyric acid (A₂bu), a neurotoxin, in tissues of flatpea ( Lathyrus sylvestris L.) necessitates a thorough understanding of the regulation of this nonprotein amino acid before the species can be recommended to livestock producers for forage applications. To determine how different concentrations and ratios of NO₃ and NH⁺₄ in growth media influence the levels of A₂bu and other free amino acids in the 'Lathco'flatpea cultivar, plants were grown hydroponically in controlled environments. The concentration of A₂bu was highest in tissues when the NO₃ to NH⁺₄ ratio in the nutrient solution was low. Responses of amides and other nonprotein amino acids, especially in the roots, followed a similar trend. Free protein amino acids in leaves and stems were generally unaffected by changes in NO₃ to NH⁺₄ ratios. In roots, protein amino acids increased as the NO₃ to NH⁺₄ ratio in the growth medium increased. Ammonium inhibited shoot and root growth; NO₃ alleviated the toxic effects of NH⁺₄. Soluble protein concentrations were higher in the shoots of NO₃-fed plants and in the roots of plants supplied with NH⁺₄. These results suggest that accumulation of A₂bu and other nonprotein amino acids, as well as asparagine and glutamine, plays a role in detoxification of NH⁺₄ and storage of N.     

The ruminant dental grooming apparatus

Correlations between dental morphology and dietary preferences in ruminants explain the similarities but not the differences in relative incisor width encountered within the group. Observations on African browsing antelope have revealed extensive use of the lateral anterior dental elements for grooming purposes using a distinctive upward sweeping movement of the head. Inspection of these dental elements (I₂, I₃₎ and C) reveals a comb-like array remarkably similar to the prosimian tooth-comb. An hypothesis is presented to explain differences in incisor morphology based on the use of the teeth for purposes other than eating. The alternative biological role has implications for the use of dental characteristics in the determination of the feeding ecology of living and extinct ruminants.