Abscisic Acid-Induced Membrane Potential Changes in Barley Aleurone Protoplasts: a Possible Relevance for the Regulation of rab Gene Expression

The effect of ABA on the membrane potential of barley (Hordeumvulgare cv. Himalaya) aleurone protoplasts was studied by measuringthe distribution of the lipophilic cation tetraphenylphosphonium(TPP⁺). The resting membrane potential (E_m) according to ourmeasurements with TPP⁺ is about –53 mV and is in agreementwith membrane potential values as measured with intracellularmicroelectrodes (about –55 mV). The TPP⁺-measurementscould demonstrate a clear dependence of the resting E_m on theexternal pH (pH_e). Stimulation of the protoplasts with ABA induced a transienthyperpolarization of the membrane to –62 mV as measuredwith TPP⁺. The hyperpolarization was ABA-concentration dependent. Inhibition of the H⁺-ATPases with the specific proton pump inhibitorsdiethylstilbestrol (DES) or Micanozole effectively preventedhyperpolarization. This indicates that the hyperpolarizationis consistent with the activation of plasma membrane H⁺-ATPases.The K⁺-inward rectifier inhibitor BaCl₂ was able to prolongthe hyperpolarization. This result suggests that the hyperpolarizationcauses the opening of K⁺-channels. The ABA-induced proton-pump activation may be involved in ABA-inducedgene-expression, as DES was able to inhibit this gene expression.BaCl₂ did only show a slight inhibitory effect on ABA-inducedgene-expression. (Received January 4, 1994; Accepted April 12, 1994)     

Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts

Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.     

Selective Expansion of Chimeric Antigen Receptor-targeted T-cells with Potent Effector Function using Interleukin-4

Polyclonal T-cells can be directed against cancer using transmembrane fusion molecules known as chimeric antigen receptors (CARs). Although preclinical studies have provided encouragement, pioneering clinical trials using CAR-based immunotherapy have been disappointing. Key obstacles are the need for robust expansion ex vivo followed by sustained survival of infused T-cells in patients. To address this, we have developed a system to achieve selective proliferation of CAR⁺ T-cells using IL-4, a cytokine with several pathophysiologic and therapeutic links to cancer. A chimeric cytokine receptor (4αβ) was engineered by fusion of the IL-4 receptor α (IL-4Rα) ectodomain to the β_c subunit, used by IL-2 and IL-15. Addition of IL-4 to T-cells that express 4αβ resulted in STAT3/STAT5/ERK phosphorylation and exponential proliferation, mimicking the actions of IL-2. Using receptor-selective IL-4 muteins, partnering of 4αβ with γ_c was implicated in signal delivery. Next, human T-cells were engineered to co-express 4αβ with a CAR specific for tumor-associated MUC1. These T-cells exhibited an unprecedented capacity to elicit repeated destruction of MUC1-expressing tumor cultures and expanded through several logs in vitro. Despite prolonged culture in IL-4, T-cells retained specificity for target antigen, type 1 polarity, and cytokine dependence. Similar findings were observed using CARs directed against two additional tumor-associated targets, demonstrating generality of application. Furthermore, this system allows rapid ex vivo expansion and enrichment of engineered T-cells from small blood volumes, under GMP-compliant conditions. Together, these findings provide proof of principle for the development of IL-4-enhanced T-cell immunotherapy of cancer.     

Rapid susceptibility testing and microcolony analysis of Candida spp. cultured and imaged on porous aluminum oxide

Background

Acquired resistance to antifungal agents now supports the introduction of susceptibility testing for species-drug combinations for which this was previously thought unnecessary. For pathogenic yeasts, conventional phenotypic testing needs at least 24 h. Culture on a porous aluminum oxide (PAO) support combined with microscopy offers a route to more rapid results.

Methods

Microcolonies of Candida species grown on PAO were stained with the fluorogenic dyes Fun-1 and Calcofluor White and then imaged by fluorescence microscopy. Images were captured by a charge-coupled device camera and processed by publicly available software. By this method, the growth of yeasts could be detected and quantified within 2 h. Microcolony imaging was then used to assess the susceptibility of the yeasts to amphotericin B, anidulafungin and caspofungin (3.5 h culture), and voriconazole and itraconazole (7 h culture).

Significance

Overall, the results showed good agreement with EUCAST (86.5% agreement; n = 170) and E-test (85.9% agreement; n = 170). The closest agreement to standard tests was found when testing susceptibility to amphotericin B and echinocandins (88.2 to 91.2%) and the least good for the triazoles (79.4 to 82.4%). Furthermore, large datasets on population variation could be rapidly obtained. An analysis of microcolonies revealed subtle effects of antimycotics on resistant strains and below the MIC of sensitive strains, particularly an increase in population heterogeneity and cell density-dependent effects of triazoles. Additionally, the method could be adapted to strain identification via germ tube extension. We suggest PAO culture is a rapid and versatile method that may be usefully adapted to clinical mycology and has research applications.     

Modulation of germination of embryos isolated from dormant and nondormant barley grains by manipulation of endogenous abscisic acid

Dormant and non-dormant barley (Hordeum distichum L.) grains with identical genetic backgrounds were obtained by maturing grains under different climate conditions. When isolated embryos from dormant grains were incubated in a well containing a fixed volume of water (300 l), the germination rate and percentage were dependent on the embryo number per well. A higher embryo number per well was correlated with a lower germination rate and percentage. However, this was not the case for the embryos isolated from nondormant grains. During germination, the endogenous cis-abscisic acid (ABA) in isolated embryos from both dormant and nondormant grains was analyzed. The inhibitory effect on germination of a higher number per well of isolated dormant embryos was due to diffusion of endogenous ABA out of the embryos and accumulation of ABA in the incubation medium. Moreover, there was de-novo synthesis of ABA in embryos isolated from dormant grains during incubation but not in embryos isolated from nondormant grains. The inhibitory effect of ABA on germination of embryos isolated from dormant grains could be mimicked by addition of ABA or the medium in which dormant embryos had been placed. Embryos isolated from nondormant grains were insensitive to addition of ABA and medium from dormant embryos. Our results demonstrate that diffusion of endogenous ABA, de-novo ABA synthesis and ABA sensitivity play a role in the control of germination. It is proposed that dormancy-breaking treatments act via changes to these processes.Abbreviations ABA cis-abscisic acid - E/W embryo(s) per well Prof. K.R. Libbenga (Institute of Molecular Plant Sciences, Leiden University) is thanked for fruitful discussions. B.V.D. was partly supported by E.E.C. BIOTECH program PL 920175.     

Molecular cloning and characterization of an inorganic pyrophosphatase from barley

A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During imbibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.     

The identification of biomarkers differentiating Mycobacterium tuberculosis and non-tuberculous mycobacteria via thermally assisted hydrolysis and methylation gas chromatography–mass spectrometry and chemometrics

Infections with non-tuberculous mycobacteria (NTM) are increasing, particularly among immune-compromised patients and those with damaged lungs. Mycobacterium tuberculosis complex (MTB) strains, however, remain the most common cause of mycobacterial infection. A rapid method of distinguishing MTB from NTM is required for correct diagnosis and tuberculosis management. We have developed an automated procedure based on thermally-assisted hydrolysis and methylation followed by gas chromatography–mass spectrometry (THM–GC–MS) and advanced chemometrics to differentiate MTB from NTM. We used early cultures of mycobacteria in this first step towards the direct identification of these bacteria in sputum using a hand-held portable device. To build a classification model, we used 44 strains including 15 MTB and 29 NTM. A matrix of the aligned dataset containing ~45,700 features (retention time/mass pairs) for the 44 observations was submitted to partial least squares discriminant analysis (PLS–DA). We could reduce the number of features down to 250 without compromising the accuracy of the model. Twenty different compounds were found through mass spectral interpretation of these 250 features. Some of these compounds have not been linked to tuberculosis before, others have been proposed previously as diagnostic biomarkers for this disease. We have built a final model based on our proposed biomarkers that performed with 95 % accuracy in distinguishing MTB from NTM in early cultures. Since all these biomarkers have been chemically identified, work can proceed towards the development of simpler, bed-side diagnostic tests to differentiate MTB from NTM in sputum.     

Development and Validation of a Prediction Model for Tube Feeding Dependence after Curative (Chemo-) Radiation in Head and Neck Cancer

Background

Curative radiotherapy or chemoradiation for head and neck cancer (HNC) may result in severe acute and late side effects, including tube feeding dependence.The purpose of this prospective cohort study was to develop a prediction model for tube feeding dependence 6 months (TUBE_M6) after curative (chemo-) radiotherapy in HNC patients.

Patients and Methods

Tube feeding dependence was scored prospectively. To develop the multivariable model, a group LASSO analysis was carried out, with TUBE_M6 as the primary endpoint (n = 427). The model was then validated in a test cohort (n = 183). The training cohort was divided into three groups based on the risk of TUBE_M6 to test whether the model could be extrapolated to later time points (12, 18 and 24 months).

Results

Most important predictors for TUBE_M6 were weight loss prior to treatment, advanced T-stage, positive N-stage, bilateral neck irradiation, accelerated radiotherapy and chemoradiation. Model performance was good, with an Area under the Curve of 0.86 in the training cohort and 0.82 in the test cohort. The TUBE_M6-based risk groups were significantly associated with tube feeding dependence at later time points (p<0.001).

Conclusion

We established an externally validated predictive model for tube feeding dependence after curative radiotherapy or chemoradiation, which can be used to predict TUBE_M6.     

Cytosolic alkalinization mediated by abscisic Acid is necessary, but not sufficient, for abscisic Acid-induced gene expression in barley aleurone protoplasts

We investigated whether intracellular pH (pH_i) is a causal mediator in abscisic acid (ABA)-induced gene expression. We measured the change in pH_i by a “null-point” method during stimulation of barley (Hordeum vulgare cv Himalaya) aleurone protoplasts with ABA and found that ABA induces an increase in pH_i from 7.11 to 7.30 within 45 min after stimulation. This increase is inhibited by plasma membrane H⁺-ATPase inhibitors, which induce a decrease in pH_i, both in the presence and absence of ABA. This ABA-induced pH_i increase precedes the expression of RAB-16 mRNA, as measured by northern analysis. ABA-induced pH_i changes can be bypassed or clamped by addition of either the weak acids 5,5-dimethyl-2,4-oxazolidinedione and propionic acid, which decrease the pH_i, or the weak bases methylamine and ammonia, which increase the pH_i. Artificial pH_i increases or decreases induced by weak bases or weak acids, respectively, do not induce RAB-16 mRNA expression. Clamping of the pH_i at a high value with methylamine or ammonia treatment affected the ABA-induced increase of RAB-16 mRNA only slightly. However, inhibition of the ABA-induced pH_i increase with weak acid or proton pump inhibitor treatments strongly inhibited the ABA-induced RAB-16 mRNA expression. We conclude that, although the ABA-induced the pH_i increase is correlated with and even precedes the induction of RAB-16 mRNA expression and is an essential component of the transduction pathway leading from the hormone to gene expression, it is not sufficient to cause such expression.