Physiological changes in asparagus spear tips after harvest

To extend our understanding of the physiology of asparagus after harvest, changes in respiration rate, protein and amino acid complement, and ultrastructure of tip sections (0–30 mm) of asparagus spears ( Asparagus officinalis L. cv. Limbras 10) were investigated. Spears had been stored for up to 4 days in the dark at 20°C. Respiration rate (carbon dioxide efflux) declined rapidly after harvest before stabilizing at 12 h at ca 50% of the rate at harvest. Protein, amino acid, and ammonium content of tip sections of 180 mm spears (intact tip sections) during storage, and comparable sections; excised from spears at harvest and subsequently stored (excised tip sections), were compared. Total protein content of intact and excised tip sections increased ca 10% 6–12 h after harvest, and then declined to ca 85% of harvest levels at 48 h. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed the net loss of specific proteins at 48 h. Free amino acid content of excised tip sections declined to ca 75% of harvest levels 12 h after harvest, and then increased to 150% of harvest levels by 48 h. Glutamine levels declined rapidly after harvest, and asparagine content increased ca 200% at 24 h. Similar trends in free amino acid content were found in sections of intact tips. Ammonia (ammonium ions) accumulated to ca 0.3% dry weight at 48 h in both intact and excised tip sections. Ultrastructural studies revealed that tonoplast breakdown commenced 48–96 h after harvest. Results are discussed in relation to the sequence of physiological events following harvest and the timing of mechanisms responsible for their initiation.     

Urinary 5-HIAA measurement using automated on-line solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry

Quantification of 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is useful for diagnosis and follow-up of patients with carcinoid tumors and for monitoring serotonin (5-hydroxytryptamine) metabolism in various disorders. We describe an automated method (XLC-MS/MS) that incorporates on-line solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and tandem mass spectrometric (MS/MS) detection to measure urinary 5-HIAA. Automated pre-purification of urine was carried out with HySphere-Resin GP SPE cartridges containing strong hydrophobic polystyrene resin. The analyte (5-HIAA) and internal standard (isotope-labelled 5-HIAA-d(2)) were, after elution from the cartridge, separated by reversed-phase HPLC and detected with tandem MS. Total cycle time was 5 min. 5-HIAA and its deuterated internal standard (5-HIAA-d(2)) were retained on and eluted from the SPE cartridges in high yields (81.5-98.0%). Absolute recovery was 96.5-99.6%. Intra-assay (n=20) and inter-assay (n=20) CVs for the measurement of 5-HIAA in urine in three concentration levels ranged from 0.8 to 1.4% and 1.7 to 4.2%, respectively. For urine samples from patients (n=78) with known or suspected metastatic carcinoid tumors, results obtained by XLC-MS/MS were highly correlated (R(2)=0.99) with the routinely used fluorometric method. This XLC-MS/MS method demonstrated lower imprecision and time per analysis (high-throughput) than manual solvent extraction methods and higher sensitivity and specificity than non-mass spectrometric detection techniques.     

Synthesis of redox-active ferrocene pyrazole conjugates and their cytotoxicity in human mammary adenocarcinoma MCF-7 cells

The redox-active ferrocenoyl modified pyrazole ligand (3-Fc-AMP) readily coordinates to a variety of transition metal ions. Here, we describe our findings regarding the synthesis and structural characterization of iron and cobalt complexes of Fc-AMP, and the cytotoxicity profiles of these chemicals in vitro. Using the human mammary adenocarcinoma MCF-7 cell line we show that the free ligand and three metal complexes induced cytotoxicity with calculated LC₅₀s ranging from 45.8 to 73.2 μM. The toxicity of the complexes decreased as the redox potential increased. The present study demonstrates the potential chemotherapeutic promise of metal complexes of a redox-active ferrocenoyl modified pyrazole ligand on human cancer cells.     

Functional analysis of the multi-copper oxidase from Legionella pneumophila

Multicopper oxidases have been described to have functions in copper tolerance, manganese oxidation, and iron oxidation in a range of bacteria. The putative cytoplasmic membrane multicopper oxidase from Legionella pneumophila was investigated. The mcoL gene was found to be critical for aerobic extracellular growth under either iron-limiting conditions or in the presence of ferrous Fe(II) iron, as a sole source of this essential metal. The mcoL mutants showed minor growth defects when grown in the presence of Fe(III) as the iron source. In contrast, intracellular growth and survival was not affected by the absence of the mcoL gene regardless of available iron concentration. The evidence presented here could indicate a possible role for mcoL in prevention of the toxic effects of ferrous iron during aerobic conditions. However, a function in high-affinity acquisition of iron could also be possible given the inability of the McoL mutants to grow aerobically under iron-limiting conditions.     

<Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>responds to heat shock,penicillin induced persistence,and IFN-gamma persistence by altering levels of the extracytoplasmic stress response protease HtrA

Background  

Chlamydia trachomatis, an obligate intracellular human pathogen, is the most prevalent bacterial sexually transmitted infection worldwide and a leading cause of preventable blindness. HtrA is a virulence and stress response periplasmic serine protease and molecular chaperone found in many bacteria. Recombinant purified C. trachomatis HtrA has been previously shown to have both activities. This investigation examined the physiological role of Chlamydia trachomatis HtrA.     

veA is required for toxin and sclerotial production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.     

Production of Fumonisins by Fusarium Verticillioides Strains on Solid and in a Defined Liquid Medium — Effects of L-Methionine and Inoculum

In order to evaluate the toxicological and carcinogenic effects of fumonisins, large amounts of fumonisins need to be purified, which requires optimal conditions for production in culture. Five strains of F. verticillioides were compared for their ability to produce fumonisins in solid and liquid media with and without the addition of methionine, a fumonisin precursor. Inoculations were made either with lyophilized cultures or a concentrated inoculum. Growth in liquid medium, measured by biomass, was directly correlated to total fumonisin production when a lyophilized inoculum was used. Fumonisin production was stimulated by the addition of 0.2% L-methionine to solid media for most strains. Levels ranged from 1500-3900 mg/kg in rice, and 2900-12500 mg/kg in maize cultures inoculated with lyophilized cultures; 200-4800 mg/kg in rice, and 1500-4200 mg/kg in maize cultures inoculated with concentrated inoculum. Strains that produced relatively high levels of fumonisins in solid media did not necessarily do so in liquid medium and vice versa. Total fumonisin levels in liquid medium ranged from 40-590 mg/l inoculated with lyophilized cultures and < 1-110 mg/l inoculated with concentrated inoculum, without adding the precursor. F. verticillioides strains therefore varied in their ability to produce fumonisins, and conditions for production need to be optimized individually for each strain.     

Biosynthesis and differential processing of two pools of amyloid-beta precursor protein in a physiologically inducible neuroendocrine cell

The amyloid-beta precursor protein (APP) is linked to Alzheimer's disease through its pathological proteolytic processing in the secretory pathway. Nevertheless, surprisingly little is known about the biosynthesis of endogenous APP. We therefore decided to investigate the intracellular fate of newly synthesized APP in a physiologically inducible neuroendocrine cell, the Xenopus intermediate pituitary melanotrope cell. We found that the level of both APP mRNA and protein was about threefold induced in the activated cells of black-adapted animals. Intriguingly, two pools of APP were found, only one of which was up-regulated. This induced pool became readily N- and subsequently O-glycosylated and was eventually proteolytically processed by an alpha-secretase-like cleavage event resulting in a secreted N-terminal and a cell-associated C-terminal APP fragment. Conversely, only the other (non-induced, non-glycosylated and uncleaved) pool became phosphorylated. Thus, we report on the biosynthesis of APP in a physiological context and illuminate the occurrence of two pools of APP, one of which is linked to neuroendocrine cell activation.     

The effect of surface electrical stimulation on hyolaryngeal movement in normal individuals at rest and during swallowing.

Surface electrical stimulation is currently used in therapy for swallowing problems, although little is known about its physiological effects on neck muscles or swallowing. Previously, when one surface electrode placement was used in dysphagic patients at rest, it lowered the hyolaryngeal complex. Here we examined the effects of nine other placements in normal volunteers to determine 1) whether movements induced by surface stimulation using other placements differ, and 2) whether lowering the hyolaryngeal complex by surface electrical stimulation interfered with swallowing in healthy adults. Ten bipolar surface electrode placements overlying the submental and laryngeal regions were tested. Maximum tolerated stimulation levels were applied at rest while participants held their mouths closed. Videofluoroscopic recordings were used to measure hyoid bone and subglottic air column (laryngeal) movements from resting position and while swallowing 5 ml of liquid barium, with and without stimulation. Videofluoroscopic recordings of swallows were rated blind to condition using the National Institutes of Health-Swallowing Safety Scale. Significant (P < 0.0001) laryngeal and hyoid descent occurred with stimulation at rest. During swallowing, significant (P 相似文献