An Attenuated Highly Pathogenic Chinese PRRS Viral Vaccine Confers Cross Protection to Pigs against Challenge with the Emerging PRRSV NADC30-Like Strain

A novel PRRSV strain was isolated in China that was genetically similar to the NADC30 strain which is reported to have spread throughout China. The objective of the present study was to evaluate the cross-protective efficacy of the live vaccine TJM-F92 in young pigs against challenge with a NADC30-like strain, HN201605. Twenty-five PRRSV- and antibody-free pigs were randomly divided into the following five groups: Vac/ChA, Unvac/ChA, Vac/ChB, Unvac/ChB and the mock. The pigs in groups Vac/ChA and Vac/ChB were inoculated intramuscularly with 1 mL TJM-F92 (10^5.0 TCID₅₀/mL). At 28 days post vaccination (0 days post challenge), groups Vac/ChA and Unvac/ChA were inoculated intranasally with 10^4.5 TCID₅₀/mL PRRSV strain TJ F3 (2 mL/pig), while groups Vac/ChB and Unvac/ChB were inoculated, using the same route, with the same dose of the NADC30-like strain HN201605 F3. Protective effects of the PRRSV strain were observed in all pigs in the Vac/ChA and Vac/ChB groups. Neither high fever nor signs of clinical disease were observed through the experiment in these groups, whereas pigs in Unvac/ChA group exhibited serious clinical symptoms, pathological lesions, and weight loss. In Unvac/ChB group, pigs developed milder clinical symptoms, which demonstrated that the NADC30-like strain HN201605 had moderate pathogenicity. The results suggest that the MLV vaccine strain TJM-F92 is an effective and safe vaccine candidate for use in China.     

Molecular cloning,purification, and characterization of a novel thermostable cinnamoyl esterase from Lactobacillus helveticus KCCM 11223

A gene encoding cinnamoyl esterase (CE), which breaks down chlorogenic acid (ChA) into caffeic and quinic acids, was cloned from Lactobacillus helveticus KCCM 11223. The gene with an open reading frame of 759 nucleotides was expressed in Escherichia coli, which resulted in a 51.6-fold increase in specific activity compared to L. helveticus KCCM 11223. The recombinant CE exists as a monomeric enzyme having a molecular weight of 27.4?kDa. Although the highest activity was observed at pH 7, the enzyme showed stable activity at pH 4.0–10.0. Its optimum temperature was 65°C, and it also possessed a thermophilic activity: the half-life of CE was 24.4?min at 65°C. The half-life of CE was 145.5, 80.5, and 24.4?min at 60, 62, and 65°C, respectively. The K_m and V_max values for ChA were 0.153?mM and 559.6?µM/min, respectively. Moreover, the CE showed the highest substrate specificity with methyl caffeate among other methyl esters of hydroxycinnamic acids such as methyl ferulate, methyl sinapinate, methyl p-coumarate, and methyl caffeate. Ca²⁺, Cu²⁺, and Fe²⁺ significantly reduced the relative activity on ChA up to 70%. This is the first report on a thermostable CE from lactic acid bacteria that can be useful to hydrolyze ChA from plant cell walls.     

Influence of tight junctions on the interaction of salts with lingual epithelia: responses of chorda tympani and lingual nerves

The role of tight junctions in modulating responses from chorda tympani (taste) and lingual (general sensory) nerves are clarified in regard to their responses to salts. Chorda tympani (CT) responses elicited by organic sodium salts require greater Na⁺ concentrations to elicit the same magnitude of response as NaCl. These data can be understood in terms of the organic anions (compared with Cl^–) producing larger liquid-junction potentials across tight junctions between taste cells which, in turn, reduces Na⁺ influx into taste cells via amiloride-inhibitable channels. The anion contribution to the CT response to different Na⁺ salts can be eliminated (or enhanced) by voltage clamping the tongue with negative (with respect to the serosal solution) potentials.Whole nerve recordings from the lingual branch of the trigeminal nerve elicited by NaCl (and other salts) were reversibly inhibited by the tight junction blocker, LaCl₃ These data suggest that small hydrophilic molecules elicit responses from trigeminal fibers by diffusing across tight junctions between epithelial cells and altering the composition of the extracellular space.     

Effect of Byproducts of Flue Gas Desulfurization on the Soluble Salts Composition and Chemical Properties of Sodic Soils

The byproducts of flue gas desulfurization (BFGD) are a useful external source of Ca²⁺ for the reclamation of sodic soils because they are comparatively cheap, generally available and have high gypsum content. The ion solution composition of sodic soils also plays an important role in the reclamation process. The effect of BFGD on the soluble salts composition and chemical properties of sodic soils were studied in a soil column experiment. The experiment consisted of four treatments using two different sodic soils (sodic soil I and sodic soil II) and two BFGD rates. After the application of BFGD and leaching, the soil soluble salts were transformed from sodic salts containing Na₂CO₃ and NaHCO₃ to neutral salts containing NaCl and Na₂SO₄. The sodium adsorption ratio (SAR), pH and electrical conductivity (EC) decreased at all soil depths, and more significantly in the top soil depth. At a depth of 0–40 cm in both sodic soil I and sodic soil II, the SAR, EC and pH were less than 13, 4 dS m⁻¹ and 8.5, respectively. The changes in the chemical properties of the sodic soils reflected the changes in the ion composition of soluble salts. Leaching played a key role in the reclamation process and the reclamation effect was positively associated with the amount of leaching. The soil salts did not accumulate in the top soil layer, but there was a slight increase in the middle and bottom soil depths. The results demonstrate that the reclamation of sodic soils using BFGD is promising.     

Tissue specificity of dopamine effects on brain ATPases

Dopamine inhibits Mg²⁺,Na⁺,K⁺- and Na⁺,K⁺-ATPase activities but does not modify Mg²⁺-ATPase activity of nerve ending membranes isolated from rat cerebral cortex. In the presence of the soluble fraction of brain, dopamine activates total, Na⁺,K⁺-, and Mg²⁺-ATPases. Dopamine stimulation of nerve ending membrane ATPases is achieved when soluble fractions of brain, kidney, or liver are used. On the other hand, dopamine effects are not observed on kidney or heart ATPase preparations. These results indicate tissue specificity of dopamine effects with respect to the enzyme source; there is no tissue specificity for the requirement of the soluble fraction to achieve stimulation of ATPases by dopamine.     

The effect of bile salts on oxygen consumption, oxidative phosphorylation and ATP-ase activity of mucosal homogenates from rat jejunum and ileum

The conjugated bile salts, sodium taurocholate and glycocholate, inhibited oxygen consumption and uncoupled oxidative phosphorylation of mucosal homogenates from rat jejunum and ileum. These bile salts also were capable of increasing the ATP-ase activity, in the presence of Na⁺ + K⁺ with Mg⁺⁺, of both mucosal homogenates. Consequently, it was concluded from the results of this investigation that the previously observed decrease in ATP levels of rat jejunum and ileum, in the presence of bile salts, can be accounted for by both a complete uncoupling of oxidative phosphorylation and by an increase in ATP-ase activity. Furthermore, the mechanism of bile salt inhibition of tissue ATP levels was discussed in relation to a regulatory role played by bile salts in the active transport of water soluble substances across the small intestine.     

Incorporation of radioactive glucosamine into macromolecules at nerve endings

Mice were injected intracerebrally with [¹⁴C]glucosamine, and incorporation into macromolecules in various subcellular fractions of brain was studied at a number of times after administration of the precursor. The [¹⁴C]glucosamine was rapidly incorporated into macromolecules of all the subcellular fractions of brain including both the soluble and particulate fractions of isolated nerve endings. Incorporation into macromolecules in the soluble fraction of nerve endings was quite extensive 3 hr after administration of the precursor and the specific acitvity of this fraction fell thereafter. In contrast there was only slight incorporation of [¹⁴C] leucine into the soluble protein from isolated nerve endings in the first few hours after administration, whereas the other subcellular fractions were maximally labelled at that time. The data suggests that, unlike protein which is largely transported to nerve endings in the axoplasm, there is extensive incorporation of carbohydrate into macromolecules in nerve endings. Whereas the protein component of a glycoprotein or mucopolysaccharide may be transported to the nerve ending from the perikaryon, the structure and function of this protein may be modified at the nerve ending by further incorporation of glucosamine, sialic acid and possibly other carbohydrates. The carbohydrate-containing macromolecules could influence nerve ending function immediately after these final synthetic reactions since these reactions occur at the nerve ending and not in the perikaryon.     

Further studies of the transport of protein to nerve endings

Mice were injected intracerebrally with [l-¹⁴C]leucine, and the specific activities of subcellular fractions of brain and effractions of isolated nerve endings were determined. There was a progressive increase in the specific activity of protein associated with isolated nerve endings after incorporation of [l-¹⁴C]leucine into whole brain protein had terminated. Although, the incorporation of [¹⁴C]leucine into soluble protein of whole brain did not differ significantly in mice which were 3 months or 1-year old, the subsequent increase in specific activity of soluble protein isolated from nerve endings was significantly greater in the younger animals; 6-month-old mice were intermediate. Therefore, changes in some aspect of the transport of protein to nerve endings is altered even after sexual maturity. Anaesthetization with pentobarbitone during incorporation of [¹⁴C]leucine into protein, and inhibition of protein synthesis with acetoxycycloheximide after incorporation of [¹⁴C]leucine was complete, did not interfere with the subsequent appearance of radioactive protein at the nerve ending. Evidence is presented for the transport, from a proximal site of synthesis, of protein associated with particulate components of the nerve ending, including synaptic vesicles.     

Cholic acid modified 2 kDa polyethylenimine as efficient transfection agent

New lipopolymers were synthesized by conjugating cholic acid (ChA) to polyethylenimines (PEI; 2 and 25 kDa) and a polyallylamine (PAA; 15 kDa) via N‐acylation to develop effective gene delivery systems. The extent of ChA substitution linearly varied with the feed ratio during synthesis, indicating good control over grafting ratio. While ChA did not affect binding to plasmid DNA (pDNA) for higher molecular weight (MW) polymers, ChA substitution to 2 kDa PEI significantly affected the pDNA binding. Toxicity of the 2 kDa PEI was unaffected by ChA substitution, but it was improved for the higher MW polymers. Using immortal 293T cells and primary cord blood‐derived mesenchymal stem cells, low MW (2 kDa) PEI was shown to display much better transfection efficiency as a result of ChA substitution, unlike the higher MW polymers. We conclude that ChA could be a suitable substituent for non‐toxic (low MW) PEIs in order to improve their transfection efficiency. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1337–1341, 2013     

Cytopathic Action of Naegleria fowleri Amoebae on Rat Neuroblastoma Target Cells

The axenically cultured, weakly pathogenic Naegleria fowleri LEE and the highly pathogenic, mouse passaged N. fowleri LEEmp are cytopathic for B103 rat nerve cells in culture. Cytopathogenicity was measured by release of radiolabeled rubidium or radiolabeled chromium from B103 target cells. Cytopathogenicity was time-dependent for up to 18 h and dependent upon amoebae effector to nerve cell target ratios of less than 1:1. Release of⁵¹ Cr from B103 cells by either LEE or LEEmp amoebae was enhanced by addition of calcium or magnesium to medium free of these divalent cations but the ion-channel inhibitor, verapamil, or the ionophore A23187 and phorbol myristate acetate did not alter release of ⁵¹ Cr from B103 cells cocultured with the amoebae. Cycloheximide or actinomycin D impaired release of ⁵¹ Cr from B103 target cells injured by either LEE or LEEmp amoebae. Both strains of amoebae were fractionated by glass bead disruption and high speed centrifugation into membrane and soluble fractions. Each fraction was incubated with either ⁸⁶Rb or ⁵¹ Cr labeled nerve cells. The membrane fraction from LEEmp was more active than the soluble fraction in facilitating rubidium and chromium release. In contrast, the soluble fraction from LEE was more active than the membrane fraction in facilitating rubidium release from radiolabeled target cells. The sequential release of ⁸⁶Rb and ⁵¹ Cr from target cells rather than the simultaneous release of the two isotopes indicates that target cell death is due to the release of ions followed later by the release of large macromolecules. The results indicate that N. fowleri amoebae injure nerve cells by two alternate mechanisms, trogocytosis or contact-dependent lysis.     

Speciation of zinc and tin(II) in dentrifices with reference to availability in saliva

Abstract

The chemical speciation of zinc- and tin(II)-containing dentrifices and of the reaction between saliva and such toothpastes has been researched using computer simulation. Pivotal formulation variables have been identified (phosphates, fluorides, citrate, etc.) and optimised to suggest formulations having maximum availability, of antimicrobial metal salts Zn²⁺ (aq) and soluble tin(II) species. Although the concept of having a single dentifrice in which both the zinc and the soluble tin(II) species are maximised together is desirable, it is concluded that at a single pH value, there cannot be maximal availability for both species, but it is possible to achieve levels of antimicrobial metal salts superior to a dentifrice containing either ingredient alone.     

Effects of supplemental nitrogen rate and source on biomass production by three weed species in fallow vegetable land

In 1983, a study was conducted to evaluate three weed species for their biomass yield potential on a fallowed tomato land with various nitrogen (N) rates and sources. Dogfennel (Eupatorium capillifolium (Lam.)Small), sida (Sida rhombifolia L.), and pigweed (Amaranthus hybridus L.), were grown with three N rates: residual, 1x and 2x (1x = 56 kg ha⁻¹ N) and with two N sources: NH₄NO₃ and isobutylidene diurea (IBDU). Sida produced the greatest fresh and dry biomass yields and percentage dry matter, 43·3, 23·3 Mg ha⁻¹ and 54·1%, respectively. Yields of the three crops increased with increasing N rates and were higher with NH₄NO₃ than with IBDU. Overall, sida at the 2x N rate with NH₄NO₃ had the highest fresh, 73·8 Mg ha⁻¹, and dry biomass, 37·9 Mg ha⁻¹, yield. Lowest residual soil total soluble salt concentration, 170 ppm, was found after pigweed, in plots which did not receive additional N. Additional N application increased residual soil total soluble salts after each of the three weeds. Thus, additional N, in NH₄NO₃ form, was necessary for high yields in a fallowed vegetable land, but the additional fertilizer also increased soil total soluble salts, regardless of the plant species.     

The effects of dopamine on Na+K+-ATPase activity in nerve ending membranes are prevented by N-ethylmaleimide

The activity of Na⁺K⁺-ATPase in the membranes of nerve endings isolated from rat cerebral cortex was inhibited by dopamine. On the contrary, when the soluble fraction from cortical homogenates was added, dopamine stimulated enzyme activity. By varying the concentration of the soluble fraction present in the incubation medium for Na⁺K⁺-ATPase assay, it was possible to establish that this fraction modulates those effects of dopamine on Na⁺K⁺-ATPase.The preincubation of the membranes with N-ethylmaleimide under conditions in which the Na⁺K⁺-ATPase activity was not inhibited (5 × 10^?5 M for 10 min at 37°C), prevented both the inhibitory and the stimulatory effects of dopamine observed without or with the soluble fraction from brain respectively.These results suggest that dopamine probably acts on regions of the protein containing -SH groups, different from those sites responsible for the catalytic activity of the enzyme.     

Cholesteryl hemiazelate identified in CVD patients causes in vitro and in vivo inflammation

Oxidation of PUFAs in LDLs trapped in the arterial intima plays a critical role in atherosclerosis. Though there have been many studies on the atherogenicity of oxidized derivatives of PUFA-esters of cholesterol, the effects of cholesteryl hemiesters (ChEs), the oxidation end products of these esters, have not been studied. Through lipidomics analyses, we identified and quantified two ChE types in the plasma of CVD patients and identified four ChE types in human endarterectomy specimens. Cholesteryl hemiazelate (ChA), the ChE of azelaic acid (n-nonane-1,9-dioic acid), was the most prevalent ChE identified in both cases. Importantly, human monocytes, monocyte-derived macrophages, and neutrophils exhibit inflammatory features when exposed to subtoxic concentrations of ChA in vitro. ChA increases the secretion of proinflammatory cytokines such as interleukin-1β and interleukin-6 and modulates the surface-marker profile of monocytes and monocyte-derived macrophage. In vivo, when zebrafish larvae were fed with a ChA-enriched diet, they exhibited neutrophil and macrophage accumulation in the vasculature in a caspase 1- and cathepsin B-dependent manner. ChA also triggered lipid accumulation at the bifurcation sites of the vasculature of the zebrafish larvae and negatively impacted their life expectancy. We conclude that ChA behaves as an endogenous damage-associated molecular pattern with inflammatory and proatherogenic properties.     

A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris

A novel feeding strategy for enhanced protein production of hepatitis B virus surface antigen (HBsAg) in fed-batch fermentation, recombinant Pichia pastoris, has been developed. A minimal salt medium was used to grow cells in the initial batch fermentation, followed by a glycerol+salts fed-batch phase. At the end of the fed-batch phase a dry cell weight of 130 g l⁻¹ was achieved. In the absence of basal salts, the same amount of glycerol feed resulted in only 90 g l⁻¹ cell dry weight. When a limited amount of casamino acids were also included every 24 h during methanol induction, there was a two-fold increase in expression levels of HBsAg. After 192 h of induction, the expression levels of HBsAg (soluble and insoluble) reached >1 g l⁻¹ using the Mut⁻ strain. Thus, the use of basal salts in the glycerol feed, along with the addition of limited amounts of casamino acids with the methanol feed, resulted in an increased expression of total HBsAg.     

Cytolytic Activity of Naegleria fowleri Cell-free Extract1

ABSTRACT. The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30–60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by ⁵¹Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50°C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing ⁵¹Cr release from target cells by the soluble fraction of N. fowleri extracts.     

Action of some surfactants on neuromuscular transmission in frogs

The effect of bile salts, saponin, and Tween-80 on miniature end-plate potentials and electrotonic potentials of frog muscle fibers was studied. During the action of bile salts in a concentration of 10^–4 g/ml the frequency of the synaptic potentials rose sharply. Their amplitude also increased. The input resistance of the muscle fiber decreased during the action of these substances. With an increase in their concentration to 10^–3 g/ml bile salts caused an initial increase in frequency of the spontaneous synaptic potentials followed by their depression and complete disappearance. Tween-80 caused no appreciable change in synaptic activity, whereas saponin inhibited it. Lowering the external calcium ion concentration by two to eight times had no influence on the stimulating effect of bile salts, but the total removal of calcium reduced it. The substances tested stimulated secretion of acetylcholine from the nerve endings, probably through changes caused in the structure of the presynaptic membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 3, pp. 305–310, May–June, 1976.     

Examination of α-exosite inhibitors against Botulinum neurotoxin A protease through structure-activity relationship studies of chicoric acid

Botulinum neurotoxins (BoNT) are among the most toxic known substances and currently there are no effective treatments for intraneuronal BoNT intoxication. Chicoric acid (ChA) was previously reported as a BoNT/A inhibitor that binds to the enzyme’s α-exosite. Herein, we report the synthesis and structure-activity relationships (SARs) of a series of ChA derivatives, which revealed essential binding interactions between ChA and BoNT/A. Moreover, several ChA-based inhibitors with improved potency against the BoNT/A were discovered.     

Biotransformation and improved enzymatic extraction of chlorogenic acid from coffee pulp by filamentous fungi

The highest enzymatic extraction of covalent linked chlorogenic (36.1%) and caffeic (CA) (33%) acids from coffee pulp (CP) was achieved by solid‐state fermentation with a mixture of three enzymatic extracts produced by Aspergillus tamarii, Rhizomucor pusillus, and Trametes sp. Enzyme extracts were produced in a practical inexpensive way. Synergistic effects on the extraction yield were observed when more than one enzyme extract was used. In addition, biotransformation of chlorogenic acid (ChA) by Aspergillus niger C23308 was studied. Equimolar transformation of ChA into CA and quinic acids (QA) was observed during the first 36 h in submerged culture. Subsequently, after 36 h, equimolar transformation of CA into protocatechuic acid was observed; this pathway is being reported for the first time for A. niger. QA was used as a carbon source by A. niger C23308. This study presents the potential of using CP to produce enzymes and compounds such as ChA with biological activities. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 337–345, 2013     

New glucuronoglucans obtained by oxidation of amylose at position 6

By adding finely powdered sodium nitrite to solutions of amylose in 85% (w/w) orthophosphoric acid, a series of d-glucurono-d-glucans was prepared, having number-average molecular weights of ∼10⁴ and GlcA/Glc ratios varying from 0.5 to 3.0. The products were soluble in water, both as sodium salts and free acids, formed complexes with iodine having λ_max in the range of 508–574 nm, and exhibited strongly pH-dependent chiroptical properties.