Rapid Chromatic Detection of Bacteria by Use of a New Biomimetic Polymer Sensor

We present a new platform for visual and spectroscopic detection of bacteria. The detection scheme is based on the interaction of membrane-active compounds secreted by bacteria with agar-embedded nanoparticles comprising phospholipids and the chromatic polymer polydiacetylene (PDA). We demonstrate that PDA undergoes dramatic visible blue-to-red transformations together with an intense fluorescence emission that are induced by molecules released by multiplying bacteria. The chromatic transitions are easily identified by the naked eye and can also be recorded by conventional high-throughput screening instruments. Furthermore, the color and fluorescence changes generally occur in shorter times than the visual appearance of bacterial colonies on the agar. The chromatic technology is generic and simple, does not require identification a priori of specific bacterial recognition elements, and can be applied for detection of both gram-negative and gram-positive bacteria. We demonstrate applications of the new platform for reporting on bacterial contaminations in foods and for screening for bacterial antibiotic resistance.     

Galectin-1 is a novel structural component and a major regulator of h-ras nanoclusters

The organization of Ras proteins into nanoclusters on the inner plasma membrane is essential for Ras signal transduction, but the mechanisms that drive nanoclustering are unknown. Here we show that epidermal growth factor receptor activation stimulates the formation of H-Ras.GTP-Galectin-1 (Gal-1) complexes on the plasma membrane that are then assembled into transient nanoclusters. Gal-1 is therefore an integral structural component of the H-Ras-signaling nanocluster. Increasing Gal-1 levels increases the stability of H-Ras nanoclusters, leading to enhanced effector recruitment and signal output. Elements in the H-Ras C-terminal hypervariable region and an activated G-domain are required for H-Ras-Gal-1 interaction. Palmitoylation is not required for H-Ras-Gal-1 complex formation, but is required to anchor H-Ras-Gal-1 complexes to the plasma membrane. Our data suggest a mechanism for H-Ras nanoclustering that involves a dual role for Gal-1 as a critical scaffolding protein and a molecular chaperone that contributes to H-Ras trafficking by returning depalmitoylated H-Ras to the Golgi complex for repalmitoylation.     

Identification of Galacturonic Acid-1-phosphate Kinase, a New Member of the GHMP Kinase Superfamily in Plants, and Comparison with Galactose-1-phosphate Kinase

The process of salvaging sugars released from extracellular matrix, during plant cell growth and development, is not well understood, and many molecular components remain to be identified. Here we identify and functionally characterize a unique Arabidopsis gene encoding an α-d-galacturonic acid-1-phosphate kinase (GalAK) and compare it with galactokinase. The GalAK gene appeared to be expressed in all tissues implicating that glycose salvage is a common catabolic pathway. GalAK catalyzes the ATP-dependent conversion of α-d-galacturonic acid (d-GalA) to α-d-galacturonic acid-1-phosphate (GalA-1-P). This sugar phosphate is then converted to UDP-GalA by a UDP-sugar pyrophosphorylase as determined by a real-time ¹H NMR-based assay. GalAK is a distinct member of the GHMP kinase family that includes galactokinase (G), homoserine kinase (H), mevalonate kinase (M), and phosphomevalonate kinase (P). Although these kinases have conserved motifs for sugar binding, nucleotide binding, and catalysis, they do have subtle difference. For example, GalAK has an additional domain near the sugar-binding motif. Using site-directed mutagenesis we established that mutation in A368S reduces phosphorylation activity by 40%; A41E mutation completely abolishes GalAK activity; Y250F alters sugar specificity and allows phosphorylation of d-glucuronic acid, the 4-epimer of GalA. Unlike many plant genes that undergo duplication, GalAK occurs as a single copy gene in vascular plants. We suggest that GalAK generates GalA-1-P from the salvaged GalA that is released during growth-dependent cell wall restructuring, or from storage tissue. The GalA-1-P itself is then available for use in the formation of UDP-GalA required for glycan synthesis.d-Galacturonic acid (d-GalA)³ is a quantitatively major glycose present in numerous plant polysaccharides including pectins and arabinogalactan proteins (1, 2). The synthesis of these polysaccharides requires a large number of glycosyltransferases and diverse nucleotide-sugar (NDP-sugar) donors (1, 3). Some of these NDP-sugars are formed by interconversion of pre-existing NDP-sugars and by salvage pathways. For example, the main pathway for UDP-GalA formation is the 4-epimerization of UDP-GlcA, a reaction catalyzed by UDP-GlcA 4-epimerase (4–6). However, in ripening Fragaria fruit d-GalA is incorporated into pectin (7). It is likely that a sugar kinase converts the d-GalA to GalA-1-P (8), which is then converted to UDP-GalA by a nonspecific UDP-sugar pyrophosphorylase (9). Myo-inositol may also be a source of GalA for polysaccharide biosynthesis (10).Galacturonic acid is likely to be generated by enzyme-catalyzed hydrolysis of pectic polysaccharides in plant tissues. Polysaccharide hydrolase activities are present in germinating seeds (11, 12), in germinating and elongating pollen (13–15), and in ripening fruit (14). Thus, monosaccharide salvage pathways may be required for normal plant growth and development.Numerous sugar-1-P kinases, including d-Gal-1-P kinase (16), l-Ara-1-P kinase (17), and l-Fuc-1-P kinase (18), have been described (19), but no d-GalA-1-P kinase has been identified in any species to account for the hydrolysis and recycle of pectic polymers. The subsequent pyrophosphorylation of UDP-sugars could be carried out by UDP-sugar pyrophosphorylases (20).Here, we report the identification and characterization of a functional galacturonic acid kinase (GalAK). We compared the activity of GalAK with a previously uncharacterized Arabidopsis GalK and discussed the evolution of these sugar kinase members of the GHMP kinase.     

Proton-sensing Ca2+ binding domains regulate the cardiac Na+/Ca2+ exchanger

The cardiac Na(+)/Ca(2+) exchanger (NCX) regulates cellular [Ca(2+)](i) and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C(2) regulatory domains, Ca(2+) binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, I(NCX), whereas [H(+)](i) was varied using an ammonium chloride "rebound" method at constant extracellular pH 7.4. At pH(i) = 7.2 and [Ca(2+)](i) < 120 nM, I(NCX) was less than 4% that of its maximally Ca(2+)-activated value. I(NCX) increases steeply at [Ca(2+)](i) between 130-150 nM with a Hill coefficient (n(H)) of 8.0 ± 0.7 and K(0.5) = 310 ± 5 nM. At pH(i) = 6.87, the threshold of Ca(2+)-dependent activation of I(NCX) was shifted to much higher [Ca(2+)](i) (600-700 nM), and the relationship was similarly steep (n(H) = 8.0±0.8) with K(0.5) = 1042 ± 15 nM. The V(max) of Ca(2+)-dependent activation of I(NCX) was not significantly altered by low pH(i). The Ca(2+) affinities for CBD1 (0.39 ± 0.06 μM) and CBD2 (K(d) = 18.4 ± 6 μM) were exquisitely sensitive to [H(+)], decreasing 1.3-2.3-fold as pH(i) decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C(2) regulatory domains CBD1 and CBD2.     

Acoustic and optical transduction of BuChE binding to procainamide modified surfaces

A novel polymer, poly(procainamide), PPA, containing numerous binding sites for cholinesterases was synthesized as a recognition layer for butyryl cholinesterase (BuChE) interaction with the ligand procainamide, utilizing TSM and SPR sensors. The polymer was synthesized by the reaction of methacryloyl chloride and procainamide followed by radical polymerization. Sensor surfaces (Au or SiO(2)) were spin-coated by the polymer solution to form thin layers. Binding of BuChE was found to be sensitive to the drying procedure of the polymer layer. The binding of BuChE to the polymer coated sensors was monitored on-line by following the response of thickness shear mode (TSM) and surface plasmon resonance (SPR) sensors. Binding of BuChE to PPA-coated TSM sensors were shown to follow a Langmuir isotherm giving association constant 3.4x10(6) M(-1).     

Bulgarian Jews in Israel: genetic blood markers. Red-cell antigens, serum proteins and red-cell isozymes

Two hundred and sixteen unrelated Bulgarian Jews were typed for the following genetic systems: ABO, MNS, Rh, Kell and Duffy of the blood groups; ADA, AK1, ACP1, ESD, GLO, PGD, PGM1 and PGM2 of the red-cell enzymes, and for the serum proteins HP, GC and PI. A comparison of observed gene frequencies with those of two other Sephardi Jewish groups, from Libya and Morocco, disclosed significant heterogeneity in several systems. This was mostly due to Moroccan Jews differing from Bulgarian or from both the Libyan and Bulgarian Jews. A comparison of gene frequencies in Bulgarian Jews with those in Oriental Jews from Iraq and in Ashkenazi Jews from Poland disclosed a similarity between the three groups in Rh, ADA, GLO, PGM1 and HP. The frequencies for the above systems in the three groups were closer to those of Middle Easterners than to those of Europeans. A different pattern was observed for GC and PI, in which Bulgarian resembled Polish Jews and differed significantly from Iraqi Jews. This probably reflects an outcome of convergent adaptive processes.     

Ecological correlates between cladocerans and their endoparasites from permanent and rain pools: patterns in community composition and diversity

Water fleas (Cladocera) constitute a major component in freshwater food webs, with important ecosystem-level consequences. Their abundance and richness are strongly influenced by their ecology and coevolution with numerous endoparasites. We investigated how parasitism shapes cladoceran community structure and diversity. We surveyed 204 freshwater permanent and rain pools in Israel, identified all cladoceran specimens and screened them for infection. Daphniid species richness in this survey was lower than in previous surveys and the distribution pattern of the species was different, most likely due to local extinction and habitat loss. We recorded a total of 21 taxa of endoparasites, of which 13 are most likely species not yet described. Variation in parasite richness among hosts and sites could not be attributed to differences in host body size and behavioral feeding strategies. We extend the known host range and geographic distribution of eight parasites from Europe and North America (between latitudes 40° and 70°) to much southern areas (latitudes 31° and 32°) and to different climate zones (arid and semi-arid areas). In many infected populations we found co-occurrence of at least two endoparasites, and in most of these cases Daphnia individuals were found to be infected by several endoparasite species simultaneously. Such multiple infections may have important consequences for community structure as well as host?Cparasite coevolution.     

Ovarian hormones modulate 'compulsive' lever-pressing in female rats

Life events related to the female hormonal cycle may trigger the onset of obsessive-compulsive disorder (OCD) or exacerbate symptoms in women already suffering from it. These observations suggest a possible role for ovarian hormones in the course of this disorder. Yet, the mechanisms that may subserve the modulatory effect of ovarian hormones are currently unknown. The aim of the present study was therefore to test the role of ovarian hormones in the signal attenuation rat model of OCD. Experiment 1 compared the behavior of pre-pubertal and adult male and female rats in the model, and found no age and sex differences in compulsive responding. Experiment 2 found that compulsive responding fluctuates along the estrous cycle, being highest during late diestrous and lowest during estrous. Acute administration of estradiol to pre-pubertal female rats was found to attenuate compulsive behavior (Experiment 3), and withdrawal from chronic administration of estradiol was shown to increase this behavior (Experiment 4). These findings extend the use of the signal attenuation model of OCD to female rats, and by demonstrating that the model is sensitive to the levels of ovarian hormones, provide the basis for using the model to study the role of ovarian hormones in OCD. In addition, the present findings support the hypothesis that the increased risk of onset and exacerbation of OCD in women post-partum may be a result of the decrease in the level of estradiol, which was elevated during pregnancy.