A stylar ribonuclease assay to detect self-compatible seedlings in almond progenies

Six almond progenies, each the product of a cross between a self-compatible and a self-incompatible parent, were analysed for stylar ribonucleases. Proteins were extracted and separated using non-equilibrium pH gradient electrofocusing (NEPHGE), and the gels were stained for ribonuclease activity. Most seedlings showed either two principal bands, interpreted as corresponding to two incompatibility alleles, or a single band. The seedlings were also bagged in the field at flowering time to determine fruit set after selfing, and some were also examined for the growth of pollen-tubes in selfed styles using UV fluorescence microscopy. With very few exceptions, those seedlings showing single-banded zymograms were found to be self-compatible according to field and microscope studies, and those with two bands were found to be self-incompatible. We conclude that the allele for self-compatibility in almond does not code for ribonuclease activity and that the ribonuclease isoenzyme assay is a convenient technique for predicting self-compatibility in segregating progenies. A novel band in two derivatives of ’Ferrastar’ was ascribed to a new incompatibility allele, S ₁₀ . Received: 19 January 1999 / Accepted: 30 January 1999     

Chemical selection of mutants that affect ADH activity in Drosophila. III. Effects of ethanol

A chemical selection scheme is presented for the isolation of rare Adh-positive Drosophila. It makes use of the fact that flies lacking detectable ADH activity die as adults or larvae on relatively low concentrations of ethanol in the medium. We have demonstrated that this procedure is a practical one by crossing two Adh-negative alleles, screening 1.5×10⁶ embryos, and isolating 14 Adh-positive survivors.Supported by Postdoctoral Fellowship GM-57 from the National Institutes of Health to C. V. and by Grant GM-18254 from the NIH.Contribution No. 841 from the Department of Biology, The Johns Hopkins University.     

A new effective assay to detect antimicrobial activity of filamentous fungi

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time.     

Critical view on the monochlorodimedone assay utilized to detect haloperoxidase activity

The current study aimed to identify the halogenating enzymes involved in the biosynthesis of the ambigols A, B, C and tjipanazole D, isolated from the cyanobacterium Fischerella ambigua. Haloperoxidase (HPO) activity within F. ambigua was therefore assayed spectrophotometrically by using monochlorodimedone (MCD) during protein purification. This strategy revealed the isolation of a protein positive in the MCD-assay, but an involvement in halogenating processes could not be verified. N-terminal sequencing rather demonstrated homology to cytochrome c(6) from other cyanobacteria and green algae. From our findings it thus has to be concluded that the spectrophotometrical MCD-assay routinely used to detect HPO activity may yield false positive results, mainly since the assay focuses on the decline of the educt and not on the formation of the product. Our data indicate that the reaction of MCD with proteins of the cytochrome c- family leads to unspecific products.     

Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings

The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13, 15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of detergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween 20, Brij 35, Dobanol, n-dodecylglucoside) above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added. This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples.     

Analysis of Arabidopsis genes encoding putative class III lipases

Triacylglycerol lipases are class of enzymes which catalyze the hydrolysis of long-chain triglycerides. They are widely found in the plant kingdom, numerous genes putatively encoding triacylglycerol lipases are sequenced but only a few of them have been characterized. Here we systematically analyzed Arabidopsis gene sequences deposited in public databases, and identified 38 putative class III lipase proteins, all of which contain a highly conserved lipase_3 domain (Pfam ID: PF01764). These 38 genes are randomly distributed on all chromosomes, and their genomic sequences consist of variable numbers of introns from zero to 13. They can be divided into four groups based on homology of protein sequences, and their potential subcellular localization is predicted to cytosol, chloroplast, mitochondria or endoplasmic reticulum. Furthermore, ten typical genes are selected to investigate their expression patterns. Most of them show weak tissue- or organ-specificity expression pattern. Several of them significantly accumulates in some tissues or organs in addition to germinated seedlings. Some of them are specifically transcribed during seed germination while others are not detected during stages of normal growth which are probably induced by stresses. In conclusion, putative Arabidopsis class III lipases display polymorphism in their sequences, gene structures and expression patterns.     

Chemicals to induce plastid signals in Arabidopsis seedlings

Abstract

Tetrapyrroles and abscisic acid (ABA) were considered as plastid signal molecules for plant cells. However, they attract hot controversies. High levels of sugars also trigger plastid signals to regulate nuclear gene expression. However, the effective concentrations of sugar are extremely high (100–10,000 times of tetrapyrroles). To resolve these controversies and inconsistencies, we investigated which chemicals at what concentrations may work as plastid signal molecules, and summarize their common chemical structures. We found that they can be classified into two categories: (1) tetrapyrroles or other carbon–nitrogen heterocyclic compounds with an adjacent three-membered ring (two of them may be incomplete), with a signaling impact order: Mg–protoporphyrin IX/ABA>Fe–protoporphyrin IX>Zn–protoporphyrin IX>protoporphyrin IX>tryptophan>histidine. The completeness of the rings with a chelated metal ion has a positive effect on the effectiveness of plastid signaling stimulation. (2) Sugar or other carbon–oxygen (sulfur) five- or six-membered ring compounds, such as cyclohexane and thiophene. The effective concentrations of the second category of signal molecules are very high (about 400 mM for the monosaccharide). The two types of signal molecules are possibly perceived by different receptors in the plastid.     

Simple luminometric assay to detect phosphoinositol-linked receptor expression in Xenopus oocytes.

A simple and sensitive method to measure the expression of phosphoinositol-linked receptors in Xenopus laevis oocytes is described. Oocytes are co-injected with the calcium photoprotein aequorin and RNA, encoding the receptor of interest. The binding of ligand to the expressed receptor increases intracellular calcium that induces the aequorin to luminesce. With an autosampler-equipped luminometer, this provides a fully automated assay of receptor expression of oocytes. This method was applied to cloning the bombesin/GRP receptor expressed in Swiss 3T3 fibroblasts. Oocytes expressing the cloned BR showed up to a 10,000-fold increase in light emission in response to bombesin. The sensitivity of this procedure allowed detection of positive luminometer signals in single oocytes injected with RNA transcribed from cDNA pools as large as 25,000 clones. These findings show the potential value of this procedure for rapid screening of expression libraries, structure/function analysis of receptors and analysis of receptor antagonists or agonists.     

Morphogenesis of rice and Arabidopsis seedlings in space.

Seedlings of rice (Oryza saliva L.) and Arabidopsis (A. thaliana L.) were cultivated for 68.5 hr in the RICE experiment on board during Space Shuttle STS 95 mission, and changes in their growth and morphology were analyzed. Microgravity in space stimulated elongation growth of both rice coleoptiles and Arabidopsis hypocotyls by making their cell walls extensible. In space, rice coleoptiles showed an inclination toward the caryopsis in the basal region and also a spontaneous curvature in the same direction in the elongating region. These inclinations and curvatures were more prominent in the Koshihikari cultivar compared to a dwarf cultivar, Tan-ginbozu. Rice roots elongated in various directions including into the air on orbit, but two thirds of the roots formed a constant angle with the axis of the caryopsis. In space, Arabidopsis hypocotyls also elongated in a variety of directions and about 10% of the hypocotyls grew into the agar medium. No clear curvatures were observed in the elongating region of Arabidopsis hypocotyls. Such a morphology of both types of seedlings was fundamentally similar to that observed on a 3 D clinostat. Thus, it was confirmed by the RICE experiment that rice and Arabidopsis seedlings perform an automorphogenesis under not only simulated but also true microgravity conditions.     

Comparison of techniques permitting to detect apoptosis in situ

Several morphological and biochemical techniques are in use for identification of apoptotic and necrotic cells in a studied cell population. It is essential to define not only the type of cell death but also to identify the apoptotic process itself, which represents a multistage, active process, requiring activation of a molecular event cascade. In the present study, we have examined and discussed effectiveness of the selected techniques detecting apoptosis in lymphocytes exposed to incubation at an elevated temperature. The appraisal involved detection of caspase-3 active form, detection of Bcl-2, TUNEL reaction, the comet assay and electrophoresis of DNA.     

ADH IB Expression,but Not ADH III,Is Decreased in Human Lung Cancer

Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer.     

A simple histochemical assay to detect cancer cells

Oxygen insensitivity of cancer cells and oxygen sensitivity of non-cancer cells in the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) activity enables detection of cancer cells in unfixed cell smears or cryostat sections of biopsies. The assay is based on reduction of the tetrazolium salt neotetrazolium. It is a cheap assay that is easy to perform. It takes only 30 min at the most. The test discriminates between adenomas and carcinomas of colon and rectum with a certainty higher than 80% and is the best prognosticator of survival of colorectal cancer patients. Pancreatic cancer can be discriminated from pancreatitis with 100% certainty. Therefore, the assay is an excellent tool for the pathologist to provide additional information in difficult cases of diagnosis of cancer and for prognosis.