Induction of ethylene synthesis and lipid peroxidation in damaged or TMV-infected tobacco leaf tissues by light

The effect of light on ethylene and ethane production in damaged leaf tissues was investigated. When whole leaves of tobacco cv. Samsun NN were damaged with liquid nitrogen, the ethylene formation was the highest, if 100?% of leaves were injured and were kept in the light, the lowest when leaves after 100?% injury were kept in darkness. Ethane production (lipid peroxidation) could be detected only in damaged, but not in control leaves, and was much higher in light than in darkness. In addition, there was a strong degradation of chlorophyll of damaged leaves kept in light. In light aminoethoxy-vinylglycine (AVG) inhibited ethylene formation in control, non-damaged whole leaves effectively, but in leaves with 100?% damage the inhibitory effect was much weaker and similar to the effect of propyl gallate (PG), a free radical scavenger. Both AVG and PG treatments decreased ethylene formation by control leaf discs and discs with 100?% damage. Ethane production was significantly inhibited by PG and slightly by AVG in the case of 100?% damage. Tiron, another free radical scavenger gave similar results on leaf discs as PG did. Paraquat (methylviologen, Pq), as a photosynthesis inhibiting and reactive oxygen species (ROS) producing herbicide produced a large amount of ethylene and ethane in light but very small amount in darkness. In accordance, tobacco mosaic virus (TMV) infection on the necrotic host resulted in significantly larger amount of ethylene and ethane formation in light than in darkness. We conclude that ethylene and ethane production of damaged plant tissues is strongly induced by light and ROS that are involved in this induction.     

Three phyla—Two type specimens—One shell: History of a snail shell revealed by modern imaging technology

In a recent revision of the gastropod genus Bellardiella, it became evident that the neotype of B. crassilabris had numerous borings over its entire shell surface. Superficial and light microscopical observation of the shell showed that the borings were made by a bryozoan belonging to the genus Penetrantia. Conventional techniques such as endocasting with the dissolution of the shell are impossible for type material. The application of non‐destructive micro‐CT‐scanning of the shell revealed the three‐dimensional arrangement of the entire colony within the shell. Careful analysis of the structure of the autozooids of the colony revealed that the species could not be assigned to any known species; we accordingly describe it as Penetrantia bellardiellae Schwaha, n. sp. The application of micro‐CT not only allowed the depiction of the new bryozoan species but also revealed the remains of a hermit crab located within the shell. The shell of Bellardiella crassilabris thus represents the type specimen of two species and contains a member of a third phylum inside. To our knowledge, this is a unique case, which also emphasizes the usefulness of micro‐CT imaging for precious specimens as well as the importance of historical museum collections.     

Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein

Currently there is limited information about the quality of immune responses elicited by candidate human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogens in primates. Here we describe a comprehensive analysis of neutralizing antibody and T-cell responses obtained in cynomolgus macaques by three selected immunization regimens. We used the previously described YU2-based gp140 protein trimers administered in an adjuvant, preceded by two distinct priming strategies: either alphavirus replicon particles expressing matched gp140 trimers or gp120 core proteins stabilized in the CD4-bound conformation. The rationale for priming with replicon particles was to evaluate the impact of the expression platform on trimer immunogenicity. The stable core proteins were chosen in an attempt to expand selectively lymphocytes recognizing common determinants between the core and trimers to broaden the immune response. The results presented here demonstrate that the platform by which Env trimers were delivered in the priming (either protein or replicon vector) had little impact on the overall immune response. In contrast, priming with stable core proteins followed by a trimer boost strikingly focused the T-cell response on the core sequences of HIV-1 Env. The specificity of the T-cell response was distinctly different from that of the responses obtained in animals immunized with trimers alone and was shown to be mediated by CD4⁺ T cells. However, this regimen showed limited or no improvement in the neutralizing antibody responses, suggesting that further immunogen design efforts are required to successfully focus the B-cell response on conserved neutralizing determinants of HIV-1 Env.     

The anticonvulsive effect of non-NMDA antagonist GYKI 52466 on 3-aminopyridine-induced primary and secondary cortical ictal activity in rat

The effect of GYKI 52466, a selective, non-competitive antagonist of the AMPA glutamate receptor subtype was investigated on the development, expression and propagation of 3-aminopyridine-induced cortical ictal activity, both in the primary and secondary focus. In one group of animals GYKI 52466 was administered intraperitoneally, 20 minutes prior to the local application of the convulsant the surface of the cortex of anaesthetized rats. Control animals were injected by physiological solution. Different parameters of electrographic ictal discharges were measured under the influence of the antagonist and compared to control values. The results demonstrate that GYKI 52466 exerts anticonvulsive effects on both the induction and the expression of primary and secondary electrical ictal activity, by delaying the onset of the first ictal period, shortening the duration of ictal activity and decreasing the amplitudes of epileptiform discharges. However, the seizure propagation was not influenced significantly. It is suggested that the initiation, maintenance and the propagation of spontaneous seizures may be controlled by separate mechanisms and that changes can occur in one of the procedures without parallel changes in others. The observations of the present study extend those reported previously by others, namely that activation of non-NMDA receptors is significantly involved in the initiation and maintenance of cortical epileptiform activity.     

Atomic resolution structures and solution behavior of enzyme-substrate complexes of Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase. Multiple conformational states and implications for the mechanism of nitroaromatic explosive degradation

The structure of pentaerythritol tetranitrate (PETN) reductase in complex with the nitroaromatic substrate picric acid determined previously at 1.55 A resolution indicated additional electron density between the indole ring of residue Trp-102 and the nitro group at C-6 of picrate. The data suggested the presence of an unusual bond between substrate and the tryptophan side chain. Herein, we have extended the resolution of the PETN reductase-picric acid complex to 0.9 A. This high-resolution analysis indicates that the active site is partially occupied with picric acid and that the anomalous density seen in the original study is attributed to the population of multiple conformational states of Trp-102 and not a formal covalent bond between the indole ring of Trp-102 and picric acid. The significance of any interaction between Trp-102 and nitroaromatic substrates was probed further in solution and crystal complexes with wild-type and mutant (W102Y and W102F) enzymes. Unlike with wild-type enzyme, in the crystalline form picric acid was bound at full occupancy in the mutant enzymes, and there was no evidence for multiple conformations of active site residues. Solution studies indicate tighter binding of picric acid in the active sites of the W102Y and W102F enzymes. Mutation of Trp-102 does not impair significantly enzyme reduction by NADPH, but the kinetics of decay of the hydride-Meisenheimer complex are accelerated in the mutant enzymes. The data reveal that decay of the hydride-Meisenheimer complex is enzyme catalyzed and that the final distribution of reaction products for the mutant enzymes is substantially different from wild-type enzyme. Implications for the mechanism of high explosive degradation by PETN reductase are discussed.     

Crystal structure of bacterial morphinone reductase and properties of the C191A mutant enzyme

The crystal structure of the NADH-dependent bacterial flavoenzyme morphinone reductase (MR) has been determined at 2.2-A resolution in complex with the oxidizing substrate codeinone. The structure reveals a dimeric enzyme comprising two 8-fold beta/alpha barrel domains, each bound to FMN, and a subunit folding topology and mode of flavin-binding similar to that found in Old Yellow Enzyme (OYE) and pentaerythritol tetranitrate (PETN) reductase. The subunit interface of MR is formed by interactions from an N-terminal beta strand and helices 2 and 8 of the barrel domain and is different to that seen in OYE. The active site structures of MR, OYE, and PETN reductase are highly conserved reflecting the ability of these enzymes to catalyze "generic" reactions such as the reduction of 2-cyclohexenone. A region of polypeptide presumed to define the reducing coenzyme specificity is identified by comparison of the MR structure (NADH-dependent) with that of PETN reductase (NADPH-dependent). The active site acid identified in OYE (Tyr-196) and conserved in PETN reductase (Tyr-186) is replaced by Cys-191 in MR. Mutagenesis studies have established that Cys-191 does not act as a crucial acid in the mechanism of reduction of the olefinic bond found in 2-cyclohexenone and codeinone.     

Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milk

AIM: To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E. coli O157) in raw and pasteurized milk. METHODS AND RESULTS: Raw milk, pasteurized milk with 1.5% fat content and pasteurized milk with 3.5% fat content were spiked with E. coli O157 at low levels. The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37 degrees C. Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFosstrade mark). Uninjured E. coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml-1 sample). Injured organisms were detected at levels of about 4 cfu 10 ml-1 sample. Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37 degrees C) and mTSBn (16 h, 37 degrees C), successively. CONCLUSIONS: The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml-1 milk sample. Injured organisms can be detected and isolated at a level almost as low as this. A resuscitation step is not recommended for the detection and isolation of injured and non-injured E. coli O157 from milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E. coli O157 requires a very sensitive method. Both methods described here are useful for such analysis.     

Combination therapy with a synthetic peptide of C-reactive protein and interleukin 2: augmented survival and eradication of pulmonary metastases

A synthetic peptide (RS-83277) derived from the structure of human C-reactive protein (CRP) was previously shown to have antitumor activity in three different murine tumor models when administered in multilamellar vesicles (MLV). The therapeutic effects were comparable to those seen with MLV-encapsulated native CRP. The present study evaluated the therapeutic and immunomodulatory effects of administering CRP peptide RS-83277 MLV simultaneously with low-dose recombinant interleukin-2 (IL-2) to C57B1/6 mice bearing established pulmonary metastases of fibrosarcoma T241. Results demonstrated that the capacity of RS-83277 MLV to inhibit tumor metastases and prolong survival was significantly augmented by combination with 10 000 U/day IL-2 i.p. Treated animals showed no evidence of toxicity. By immunohistochemistry, increased Thy 1.2⁺ cells were detectable in lungs of RS-83277 MLV/IL-2-treated animals compared to those receiving RS-83277 MLV alone. Circulating tumor necrosis factor (TNF) and interferon (IFN) were not detectable in animals receiving RS-83277 MLV alone, but TNF was significantly elevated in animals receiving IL-2. In the presence of combination therapy, however, circulating TNF was not detectable. Results suggest that the combination of synthetic CRP peptide RS-83277 MLV and low-dose IL-2 offers a therapeutic advantage over either agent alone.This work was supported in part by grant CA49950 from the National Cancer Institute (B.P.B.), and grant 43618 from the National American Cancer Society (S.D.D.)     

Detection of hepatitis B surface antigen in isolated hepatocytes.

HBsAg was detected by indirect immunofluorescent method in liver biopsy specimens of 60 symptom-free HBsAg positive volunteers. An effort was made to separate from the material intact cells suitable for studying HBsAg localization in the liver cells.