Tissue distribution of cholinesterases and anticholinesterases in native and transgenic tomato plants

Acetylcholinesterase, a major component of the central and peripheral nervous systems, is ubiquitous among multicellular animals, where its main function is to terminate synaptic transmission by hydrolyzing the neurotransmitter, acetylcholine. However, previous reports describe cholinesterase activities in several plant species and we present data for its presence in tomato plants. Ectopic expression of a recombinant form of the human enzyme and the expression pattern of the transgene and the accumulation of its product in transgenic tomato plants are described. Levels of acetylcholinesterase activity in different tissues are closely effected by and can be separated from -tomatine, an anticholinesterase steroidal glycoalkaloid. The recombinant enzyme can also be separated from the endogenous cholinesterase activity by its subcellular localization and distinct biochemical properties. Our results provide evidence for the co-existence in tomato plants of both acetylcholinesterase activity and a steroidal glycoalkaloid with anticholinesterase activity and suggest spatial mutual exclusivity of these antagonistic activities. Potential functions, including roles in plant-pathogen interactions are discussed.     

Regulation of Proton Flow and ATP Synthesis in Chloroplasts

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 mol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the and subunits of CF₁ play key roles in both regulation of activity and proton translocation.     

A dried blood spots technique based LC-MS/MS method for the analysis of posaconazole in human whole blood samples

A rugged and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method utilizing dried blood spots (DBS) was developed and validated for the analysis of posaconazole in human whole blood. Posaconazole fortified blood samples were spotted (15 μL) onto Ahlstrom Alh-226 DBS cards and dried for at least 2h. Punched spots were then extracted by using a mixture of acetonitrile and water containing stable labeled internal standard (IS). Posaconazole and its IS were separated from endogenous matrix components on a Kinetex? C18 column under gradient conditions with a mobile phase A consisting of 0.1% formic acid and a mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (70/30, v/v). The analyte and IS were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray? source operated in the positive ion mode. The assay was linear over the concentration range of 5-5000 ng/mL. The inter-run accuracy and precision of the assay were -1.8% to 0.8% and 4.0% to 10.4%, respectively. Additional assessments unique to DBS were investigated including sample spot homogeneity, spot volume, and hematocrit. Blood spot homogeneity was maintained and accurate and precise quantitation results were obtained when using a blood spot volume of between 15 and 35 μL. Human blood samples with hematocrit values ranging between 25% and 41% gave acceptable quantitation results. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible.     

Acceleration of cryo-EM Flexible Fitting for Large Biomolecular Systems by Efficient Space Partitioning

1. Download high-res image (287KB)
2. Download full-size image

     

Epidemiological trends of dermatophytoses and dermatophytes in Jerusalem between 1954 and 1981

Data for dermatophyte infections analysed for five 3-year periods between 1954 to 1981 led to the following conclusions: (1) Tinea pedis, tinea cruris and tinea manuum showed an increase in the 50's and 60's and declined in the 70's; (2) Tinea unguium and tinea corporis showed an increase during the whole period; (3) At all these sites, the percentage of Trichophyton rubrum, the main etiologic agent, increased steadily over the periods while the percentage of Trichophyton mentagrophytes, the secondary etiological agent, decreased. Epidermophyton floccosum, the third etiological agent in these sites, showed no sharp fluctuations; (4) These three dermatophytes which show similar microclimatic requirements and favour the same microecological niches, were called glabrohydrophilic. In tinea corporis they form a definite subset, their percentage being similar to that at other glabrous sites; (5) Tinea capitis was at its peak in the 50's, decreased sharply until the second half of the 70's, its main etiological agent being Trichophyton violaceum. Since 1979, an increase of tinea capitis occurred due to the newly introduced Microsporum canis; (6) Dermatophytes favouring scalp hair were called trichophilic. In tinea corporis they form a definite subset, their percentage being similar to that of tinea capitis; (7) A comparison with other studies from this country shows that macroclimate (i.e. humid warm coastal climate compared with dry cooler inland-mountain climate) is not an important factor in the etiology of tinea.     

Modified testicular expression of stress-associated "readthrough" acetylcholinesterase predicts male infertility.

Male infertility is often attributed to stress. However, the protein or proteins that mediate stress-related infertility are not yet known. Overexpression of the "readthrough" variant of acetylcholinesterase (AChE-R) is involved in the cellular stress response in a variety of mammalian tissues. Here, we report testicular overexpression of AChE-R in heads, but not tails, of postmeiotic spermatozoa from mice subjected to a transient psychological stress compared with age-matched control mice. Transgenic mice overexpressing AChE-R displayed reduced sperm counts, decreased seminal gland weight, and impaired sperm motility compared with age-matched nontransgenic controls. AChE-R was prominent in meiotic phase spermatocytes and in tails, but not heads, of testicular spermatozoa from AChE-R transgenic mice. Head-localized AChE-R was characteristic of human sperm from fertile donors. In contrast, sperm head AChE-R staining was conspicuously reduced in samples from human couples for whom the cause of infertility could not be determined, similar to the pattern found in transgenic mice. These findings indicate AChE-R involvement in impaired sperm quality, which suggests that it is a molecular marker for stress-related infertility.     

The Role of Iron-Deficiency Stress Responses in Stimulating Heavy-Metal Transport in Plants

Plant accumulation of Fe and other metals can be enhanced under Fe deficiency. We investigated the influence of Fe status on heavy-metal and divalent-cation uptake in roots of pea (Pisum sativum L. cv Sparkle) seedlings using Cd²⁺ uptake as a model system. Radiotracer techniques were used to quantify unidirectional ¹⁰⁹Cd influx into roots of Fe-deficient and Fe-sufficient pea seedlings. The concentration-dependent kinetics for ¹⁰⁹Cd influx were graphically complex and nonsaturating but could be resolved into a linear component and a saturable component exhibiting Michaelis-Menten kinetics. We demonstrated that the linear component was apoplastically bound Cd²⁺ remaining in the root cell wall after desorption, whereas the saturable component was transporter-mediated Cd²⁺ influx across the root-cell plasma membrane. The Cd²⁺ transport system in roots of both Fe-deficient and Fe-sufficient seedlings exhibited similar Michaelis constant values, 1.5 and 0.6 μm, respectively, for saturable Cd²⁺ influx, whereas the maximum initial velocity for Cd²⁺ uptake in Fe-deficient seedlings was nearly 7-fold higher than that in Fe-grown seedlings. Investigations into the mechanistic basis for this response demonstrated that Fe-deficiency-induced stimulation of the plasma membrane H⁺-ATPase did not play a role in the enhanced Cd²⁺ uptake. Expression studies with the Fe²⁺ transporter cloned from Arabidopsis, IRT1, indicated that Fe deficiency induced the expression of this transporter, which might facilitate the transport of heavy-metal divalent cations such as Cd²⁺ and Zn²⁺, in addition to Fe²⁺.     

Structure-Biological Activity Relationships in Alfalfa Antimycotic Saponins: The Relative Activity of Medicagenic Acid and Synthetic Derivatives Thereof Against Plant Pathogenic Fungi1

The antimycotic activity of medicagenic acid and of some synthetic derivatives thereof was tested against plant pathogenic fungi. In general they all possess antimycotic activity. Furthermore, in the case of Sclerotium rolfsii, compounds where the hydroxyl functions of the aglycon remained unchanged (medicagenic acid and its dimethyl ester) or could be enzymically released (3-0-β-D-glucoside of medicagenic acid dimethyl ester) were significantly more active than compounds where these functions were modified by acetylation or methylation. Selective 2-0-methylation of medicagenic acid and comparison of the antimycotic activity of the resulting derivative against S. rolfsii to that of other derivatives suggests that a potential free hydroxyl at position 3 is essential to antimycotic activity.     

Involvement of Thylakoid Overenergization in Tentoxin-Induced Chlorosis in Nicotiana spp

The purpose of this work was to clarify the mechanism of tentoxin-induced chlorosis in Nicotiana spp. seedlings. We found that chlorosis does not correlate with the inhibition of chloroplast ATP synthesis in vivo, since it occurs at tentoxin concentrations far higher than that required for the inhibition of photophosphorylation measured in the same seedlings. However, tentoxin-induced chlorosis does correlate with in vivo overenergization of thylakoids. We show that tentoxin induces overenergization in intact plants and isolated thylakoids, probably via multiple interactions with ATP synthase. Furthermore, gramicidin D, a protonophore that relieves overenergization, also relieves chlorosis. Two lines of evidence suggest that reactive oxygen species may be involved in the process of chlorosis: ascorbate, a quencher of oxygen radicals, significantly protects against chlorosis, whereas transgenic Nicotiana spp. mutants overexpressing chloroplast superoxide dismutase are partially resistant to tentoxin-induced chlorosis. It is proposed that chlorosis in developing seedlings results from overenergization of thylakoids, which leads to the generation of oxygen radicals.