Drought-induced expression of aquaporin genes in leaves of two common bean cultivars differing in tolerance to drought stress

Aquaporin proteins are part of the complex response of common bean (Phaseolus vulgaris L.) to drought which affects the quality and quantity of yield of this important crop. To better understand the role of aquaporins in common bean, drought-induced gene expression of several aquaporins was determined in two cultivars, the more drought tolerant Tiber and the less tolerant Starozagorski ?ern. The two bean cultivars were selected among 16 European genotypes based on the tolerance to drought determined by time needed for plants to wilt after withholding irrigation and yield at harvest. The expression patterns of two plasma membrane intrinsic proteins, PvPIP1;2 and PvPIP2;7, and two tonoplast intrinsic proteins, PvTIP1;1 and PvTIP4;1 in leaves of 21 day old plants were determined by RT-qPCR in both cultivars under three degrees of drought stress, and under rehydration and control conditions. Gene expression of all four examined aquaporins was down-regulated in drought stressed plants. After rehydration it returned to the level of control plants or was even higher. The responses of PvPIP2;7 and PvTIP1;1 during drought and rehydration were particularly pronounced. The gene expression of PvPIP2;7 and PvTIP4;1 during drought was cultivar specific, with greater down-regulation of these two aquaporins in drought tolerant Tiber. Under drought stress the relative water content and water potential of leaves were higher in Tiber than in Starozagorski plants. The differences in these physiological parameters indicate greater prevention of water loss in Tiber during drought, which may be associated with rapid and adequate down-regulation of aquaporins. These results suggest that the ability of plants to conserve water during drought stress involves timely and sufficient down-regulation of gene expression of specific aquaporins.     

Free Thiol Group of MD-2 as the Target for Inhibition of the Lipopolysaccharide-induced Cell Activation

MD-2 is a part of the Toll-like 4 signaling complex with an indispensable role in activation of the lipopolysaccharide (LPS) signaling pathway and thus a suitable target for the therapeutic inhibition of TLR4 signaling. Elucidation of MD-2 structure provides a foundation for rational design of inhibitors that bind to MD-2 and inhibit LPS signaling. Since the hydrophobic binding pocket of MD-2 provides little specificity for inhibitors, we have investigated targeting the solvent-accessible cysteine residue within the hydrophobic binding pocket of MD-2. Compounds with affinity for the hydrophobic pocket that contain a thiol-reactive group, which mediates covalent bond formation with the free cysteine residue of MD-2, were tested. Fluorescent compounds 2-(4′-(iodoacetamido)anilino)naphthalene-6-sulfonic acid and N-pyrene maleimide formed a covalent bond with MD-2 through Cys¹³³ and inhibited LPS signaling. Cell activation was also inhibited by thiol-reactive compounds JTT-705 originally targeted against cholesterol ester transfer protein and antirheumatic compound auranofin. Oral intake of JTT-705 significantly inhibited endotoxin-triggered tumor necrosis factor α production in mice. The thiol group of MD-2 also represents the target of environmental or endogenous thiol-reactive compounds that are produced in inflammation.TLR4 is a receptor for lipopolysaccharide (LPS)⁴ a major constituent of outer membrane of Gram-negative bacteria. MD-2 is the final LPS-binding protein in the recognition cascade before TLR4 transmits the signal across the cell membrane to activate the inflammatory response. MD-2 binds to the ectodomain of TLR4 and binds LPS either alone or in complex with TLR4 (1, 2). Mice deficient in MD-2 survive endotoxic shock (3). MD-2 has been indispensable in almost all investigated conditions of TLR4-triggered inflammation; therefore, it could represent the “Achilles'' heel” of the inflammatory response to LPS and a target for a pharmacological intervention in endotoxemia as well as other conditions involving cell activation mediated by TLR4 (4, 5). The existence of a single free cysteine residue among the seven cysteine residues has been predicted from MD-2 mutagenesis (6, 7) and molecular modeling proposed that Cys¹³³ lies in the hydrophobic pocket (8, 9). The hydrophobic binding site of MD-2 was also mapped by an apolar probe, bis-ANS, which does not contain acyl chains as most LPS antagonists yet preserves the characteristic structural motif of lipid A, consisting of a hydrophobic region and a pair of separated negatively charged groups (10). Crystal structures of MD-2 with bound eritoran or lipid IVa confirmed the location of Cys¹³³ in the hydrophobic pocket in close vicinity of bound lipid A derivatives (11, 12). The free cysteine residue inside the binding pocket can thus be a target for irreversible inhibition of MD-2 activity. An inhibitory mechanism based on a covalent modification of a free cysteine residue in the active or binding site of a protein has been demonstrated for other proteins, such as in cysteine proteases, where the cysteine residue participates in the catalytic triad (13), in cholesteryl ester transfer protein (CETP) (14), IκB kinase (15), thioredoxin reductase (16), and sortase (17).In our study, we investigated the possibility of targeting free cysteine residue of MD-2 for the inhibition of LPS signaling. We determined covalent binding into the hydrophobic pocket of MD-2 for fluorescent compounds 2-(4′-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS) and N-pyrene maleimide. Drugs JTT-705 and auranofin, already in use for alternative indications, were also shown to bind to MD-2 and decrease LPS signaling. The identity of Cys¹³³ as the residue responsible for this interaction was demonstrated by point mutagenesis. Our results confirm that the proposed mechanism of inhibition of MD-2 can have potential therapeutic value but may also have a physiological role.     

Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry

The basic requirement for probiotic bacteria to be able to exert expected positive effects is to be alive; therefore, appropriate quantification methods are crucial. Due to disadvantages of conventional microbiological methods, the bacterial quantification based on the nucleic acid detection is increasingly used. The objective of this study was to evaluate the possibility to use propidium monoazide (PMA) in combination with real-time polymerase chain reaction (PCR) method or LIVE/DEAD BacLight viability kit in combination with flow cytometry (FCM) for determination of probiotic bacteria in a lyophilised product containing Lactobacillus acidophilus LA-5 and Bifidobacterium animalis ssp. lactis BB-12. In addition, the viability of probiotic bacteria in lyophilised product during 3 months storage was investigated. In the product, the results of real-time PCR quantification of PMA-treated cells did not differ significantly from those of non-treated cells, which indicate that most of the bacterial cells retained the membrane integrity although they have lost the culturability. The results obtained by FCM analysis were comparable with those by PMA real-time PCR. In conclusion, the PMA real-time PCR and FCM determination of the viability of probiotic bacteria could complement the plate count method which considers only the culturable part of the population.     

Dissecting the thermodynamics of GAP-RhoA interactions

We describe a detailed study of the RhoA-binding epitope of the GAP domain of Graf, including the determination of the thermodynamic and kinetic parameters of the interaction of wild-type domain, and of its 15 single-site mutants, with cognate GTPases. We show that residues important for the structural integrity of the Arg-finger loop are critical for binding Rho and for the catalytic activity of GAP, but GTPase selectivity appears to be modulated by a much more subtle interplay of electrostatic and hydrophobic interactions involving residues on the periphery of the main interface. The eight residues targeted in this study are involved in three distinct patches on the surface, two of which appear to interact with highly conserved regions of the GTPase, while the third plays a role in GTPase selectivity.     

Real-time quantitative PCR assay for analysis of platelet glycoprotein IIIa gene expression

A quantitative detection assay for analysis of platelet glycoprotein GPIIIa gene expression is presented. The assay uses two fluorescently labeled TaqMan MGB probes to detect the polymorphic site in GPIIIa nucleotide sequence, leading to antigens HPA-1a and HPA-1b. In order to avoid the influence of DNA contamination on RNA quantification, a forward primer was constructed to span an exon-exon junction. The assay is therefore applicable to expression studies also in samples containing only a small amount of contaminating DNA. To standardize the amount of sample cDNA added to the reaction, amplification of endogenous control 18SrRNA was included in a separate well. The amplification validation experiment showed a high real-time PCR efficiency for HPA-1a, HPA-1b and 18SrRNA. Relative quantification was therefore performed using the comparative C(T) method. The assay was optimized on a reversely transcribed total RNA from platelets, and the specificity rate was determined by sequencing. The amount of cDNA at which amplification was still clearly detectable was 5 ng. This newly developed real-time quantitative PCR assay is a sensitive, reproducible and reliable method. It is suitable for studying different stages of megakaryopoiesis, monitoring molecular alteration in defective platelets and determining differences in the GPIIIa expression level between normal and pathological megakaryocytic differentiation pathways.     

<Emphasis Type="Italic">Mycobacterium shimoidei</Emphasis>—cavitary pulmonary disease with favorable outcome

We report a case of cavitary pulmonary disease caused by Mycobacterium shimoidei in 67-year-old female with history of asthma. Even though susceptibility testing was not available, choice of treatment regimen (streptomycin, rifampicin, ethambutol, and clarithromycin), based on a few cases with favorable outcome reported in the literature, resulted with an excellent clinical, microbiological, and radiological response. This is the first report of pulmonary disease caused by M. shimoidei, but also the first ever isolation of M. shimoidei in Croatia.     

Food patch size, food concentration and grazing efficiency of the harpacticoid Paramphiascella fulvofasciata (Crustacea, Copepoda)

Harpacticoid copepods are known as important grazers on primary producers. The underlying factors for their food selectivity and grazing efficiency are however far from well known. For instance, their patchy distribution in the marine environment is well documented but how meiofaunal organisms cope with the spatial distribution/accessibility of the available food resources is less clear.In the present study a laboratory experiment was conducted to test the grazing efficiency of Paramphiascella fulvofasciata (Copepoda, Harpacticoida) on the epipelic diatom Seminavis robusta applied in recipients of different area and in various concentrations. Diatoms were enriched in the stable isotope ¹³C in order to trace food uptake and copepods were left to graze for 4 days.We found that the grazing efficiency of P. fulvofasciata was diatom concentration-dependent. A lower diatom uptake at lower diatom densities illustrated this clear functional response. On the contrary, there was no significant effect of the area per se where the copepods could graze upon. The lack of a significant effect of area is mainly due to the high variability in uptake that was recorded in some treatments. Although P. fulvofasciata is a very motile copepod, known as endobenthic and epibenthic species, it was able to concentrate on food uptake at the bottom of the experimental unit as there was no significant difference in uptake between treatments with different water heights in the units. In addition, it was found that a diatom concentration of about 140 000 cells/cm² favours egg production of P. fulvofasciata.     

Changes in cytosolic glucose level in ATP stimulated live astrocytes

Astrocytes which lie between brain capillaries and neuronal terminals are the primary site of glucose uptake and have a key role in coupling synaptic activity to glucose utilization in the central nervous system (CNS). We used a fluorescence resonance energy transfer (FRET) based approach to monitor cytosolic glucose in astrocytes. We determined the effect of increasing extracellular glucose concentrations on FRET ratio as a measure of increased cytosolic glucose in astrocytes. By briefly raising extracellular glucose concentration, astrocytes responded promptly by increased cytosolic glucose levels, which was manifested by decreased time-dependent FRET ratio. The FRET ratio fall-time recorded at low extracellular d-glucose concentration change (from 0 to 0.5 mM) was 53 s, whereas 17 s was recorded by raising extracellular concentration of d-glucose from 0 to 10 mM, which is likely due to facilitated d-glucose entry along the increased d-glucose gradient across the plasmalemma. The relationship between the extracellular glucose concentration and the FRET ratio change is limited to the maximal ratio change, where the d-glucose plasma membrane permeability is balanced by the cytosolic utilization. We measured the effect of extracellular ATP, an important extracellular messenger for astrocyte-to-astrocyte communication, on intracellular glucose concentration. The results show that stimulation of astrocytes with ATP (1 mM) decreases cytosolic glucose concentration with a time constant of ∼145 s. The mechanism of this change is discussed.