Phytochrome action inOryza sativa L. VI. Red far-red reversible effect on early development of coleoptiles

When 3–4 mm long coleoptiles of etiolated rice seedlings (cv. Koshijiwase) were irradiated with continuous red light their growth was seriously inhibited. If a brief exposure of red light (4×10³ ergs cm⁻²) was given to the short coleoptiles, the growth rate dropped immediately after the irradiation, but the growth did not stop till the coleoptile reached some calculated length. If another brief red irradaition of the same order was given 24 hr after the first, the growth rate and the final length dropped further. The effect of red light was reversed by successively given far-red light, and this response was repeatedly red and far-red reversible. The escape reaction was rather slow so that photoreversibility was not lost at all by 8th hr, and 50% of the initial reversibility was lost within ca. 16 hr at 25±0.5 C. Blue light also induced the inhibition of coleoptile elongation, the effect was reversed by subsequent far-red irradiation, and this could be obtained repeatedly. Thus, the photoinhibition of the young coleoptile can be concluded to be under the control of phytochrome, and the mode of action appeared quite different from the previously reported results with longer coleoptiles.     

Adenylylation and catalytic properties of Mycobacterium tuberculosis glutamine synthetase expressed in Escherichia coli versus mycobacteria

Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456-464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially ( approximately 25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated ( approximately 30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.     

Carbon dioxide and poultry waste utilization for production of polyhydroxyalkanoate biopolymers by <Emphasis Type="Italic">Nostoc muscorum</Emphasis> Agardh: a sustainable approach

The new paradigm is to view wastes as resources for sustainable development. In this regard, the feasibility of poultry waste and CO₂ utilization for cultivation of a filamentous nitrogen-fixing cyanobacterium, Nostoc muscorum Agardh, was investigated for production polyhydroxyalkanoates, the biodegradable polymers. This cyanobacterium showed profound rise in biomass yield with up to 10 % CO₂ supply in airstream with an aeration rate of 0.1 vvm. Maximum biomass yield of 1.12 g L^?1 was recorded for 8 days incubation period, thus demonstrating a CO₂ biofixation rate of 0.263 g L^?1 day^?1 at 10 % (v/v) CO₂-enriched air. Poultry litter (PL) supplementation also had a positive impact on the biomass yield. The nutrient removal efficiency of N. muscorum was reflected in the significant reduction in nutrient load of PL over the experimental period. A maximum poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) [P(3HB-co-3HV)] copolymer yield of 774 mg L^?1 (65 % of dry cell wt.), the value almost 11-fold higher than the control, was recorded in 10 g L^?1 PL-supplemented cultures with 10 % CO₂ supply under the optimized condition, thus demonstrating that N. muscorum has good potential for CO₂ biomitigation and poultry waste remediation while simultaneously producing eco-friendly polymers.     

Dietary Supplementation with Organoselenium Accelerates Recovery of Bladder Expression,but Does Not Improve Locomotor Function,following Spinal Cord Injury

Selenium is an essential element required for activity of several antioxidant enzymes, including glutathione peroxidase. Because of the critical role of the antioxidant system in responding to traumatic events, we hypothesized that dietary selenium supplementation would enhance neuroprotection in a rodent model of spinal cord injury. Rats were maintained on either a control or selenium-enriched diet prior to, and following, injury. Dietary selenium supplementation, provided as selenized yeast added to normal rat chow, resulted in a doubling of selenium levels in the spinal cord. Dietary selenium reduced the time required for recovery of bladder function following thoracic spinal cord injury. However, this was not accompanied by improvement in locomotor function or tissue sparing.     

Tyrosine phosphatase STEP is a key regulator of glutamate-induced prostaglandin E2 release from neurons

The neuron-specific tyrosine phosphatase striatal-enriched phosphatase (STEP) is emerging as a key regulator of excitotoxicity, which is involved in the pathogenesis of both acute and chronic neurological diseases. However, the intracellular mechanisms that are regulated by STEP to confer neuroprotection against excitotoxic insults are not well understood. The present study investigates the role of STEP in regulating neuronal release of the proinflammatory prostanoid prostaglandin E₂ (PGE₂), which is associated with a wide range of pathological conditions. The findings show that glutamate-mediated activation of the N-methyl-D-aspartic acid receptor in STEP-deficient neurons leads to rapid and sustained increase in the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), a signaling molecule involved in the production of inflammatory mediators. Such sustained p38 MAPK activation increases the activity of cytosolic phospholipase A₂, which catalyzes the release of arachidonic acid, the initial substrate for PGE₂ biosynthesis. Sustained p38 MAPK activation also induces nuclear factor-κB–mediated increase in expression of cyclooxygenase-2 that is involved in the conversion of arachidonic acid to prostanoids, resulting in enhanced biosynthesis and release of PGE₂ from neurons. Restoration of STEP function with a STEP mimetic (TAT-STEP-myc peptide) significantly decreases the activation of p38 MAPK–mediated cytosolic phospholipase A₂/cyclooxygenase-2/PGE₂ signaling cascade. This study identifies an important mechanism involved in the neuronal release of the proinflammatory mediator PGE₂ after excitotoxic insult and highlights for the first time the immunomodulatory ability of a neuronal tyrosine phosphatase.     

Escherichia coli O157:H7 Strain Origin,Lineage, and Shiga Toxin 2 Expression Affect Colonization of Cattle

Enterohemorrhagic Escherichia coli O157:H7 has evolved into an important human pathogen with cattle as the main reservoir. The recent discovery of E. coli O157:H7-induced pathologies in challenged cattle has suggested that previously discounted bacterial virulence factors may contribute to the colonization of cattle. The objective of the present study was to examine the impact of lineage type, cytotoxin activity, and cytotoxin expression on the amount of E. coli O157:H7 colonization of cattle tissue and cells in vitro. Using selected bovine- and human-origin strains, we determined that lineage type predicted the amount of E. coli O157:H7 strain colonization: lineage I > intermediate lineages > lineage II. All E. coli O157:H7 strain colonization was dose dependent, with threshold colonization at 10³ to 10⁵ CFU and maximum colonization at 10⁷ CFU. We also determined that an as-yet-unknown factor of strain origin was the most dominant predictor of the amount of strain colonization in vitro. The amount of E. coli O157:H7 colonization was also influenced by strain cytotoxin activity and the inclusion of cytotoxins from lineage I or intermediate lineage strains increased colonization of a lineage II strain. There was a higher level of expression of the Shiga toxin 1 gene (stx₁) in human-origin strains than in bovine-origin strains. In addition, lineage I strains expressed higher levels of the Shiga toxin 2 gene (stx₂). The present study supports a role for strain origin, lineage type, cytotoxin activity, and stx₂ expression in modulating the amount of E. coli O157:H7 colonization of cattle.Enterohemorrhagic Escherichia coli O157:H7 is a bacterium that causes serious human disease outbreaks through the consumption of contaminated food or water (39). Mature cattle are considered the primary reservoir for E. coli O157:H7 and historically were reported to have no symptoms or pathologies (17, 23, 38); this was attributed both to a lack of receptors for a critical E. coli O157:H7 virulence factor, Shiga toxin 1 (Stx1 [29]), and to a differential expression of type III protein secretion system effector molecules such as EspA, EspD, and Iha (25, 30) in cattle compared to humans. In 2008, it was established for the first time that E. coli O157:H7 causes mild to severe intestinal pathology in persistent shedding cattle (5, 26) and that the secreted cytotoxins enhanced E. coli O157:H7 colonization of intestinal tissues of cattle (6). This suggested that cattle were susceptible to E. coli O157:H7 infection and that previously discounted virulence factors could influence the amount of colonization in cattle.Three distinct E. coli O157:H7 lineages have been identified based on the lineage specific polymorphism assay (LSPA-6) that suggests both the evolutionary history of the strain and their propensity to be present among animals, the environment, and clinical human isolates (21, 22, 24, 33, 40, 42). Typically, two predominant lineages have been described, lineages I and II (22, 40) and, more recently, intermediate lineages that have characteristics of lineage I and/or II have been reported at higher frequency among cattle (34). Although all E. coli O157:H7 lineages have been isolated from feedlot cattle, the predominant recovery of lineage I from clinical human illnesses suggests that this particular lineage type has unique expression patterns that may contribute to its preferential colonization of humans. There is some evidence to suggest that lineage I strains do not express certain virulence factors in bovine hosts, whereas other factors such as cytotoxins are expressed equally irrespective of host (30). One virulence factor associated with all lineages is the bacterium''s ability to form intimate attaching-and-effacing lesions or colonization sites in the ilea of susceptible animals (28). The amount of colonization is enhanced by the expression of Shiga toxin 2 (Stx2) through both an increase in the expression of alternative non-TIR (translocated intimin receptor) colonization sites (31) and toxicity to the absorptive epithelial cells (32). In cattle, attaching-and-effacing lesions are also formed (5), and Stx2 increases colonization but is not cytotoxic to epithelial cells from the jejuna and descending colons of cattle (4). Differential expression of stx₂ among E. coli O157:H7 lineages is also linked to the increased pathogenicity of lineage I strains in humans (25), and this may affect cattle similarly. Together, this information suggests that at least some similar virulence factors affecting E. coli O157:H7 colonization in humans also function in cattle.In order to gain a better understanding of the factors modulating E. coli O157:H7 colonization in cattle, we compared the ability of lineage I, lineage II, and intermediate lineages isolated from human sources to colonize the jejunum tissue and a colonic cell line from cattle. We hypothesized that the bovine colonic cell line could be used as a model system to reflect E. coli O157:H7 colonization of tissue. To confirm the value of this model, the role of strain origin in colonization of cattle was examined. In order to understand the differences in colonization associated with lineage and strain origins, we assessed cytotoxin expression, secreted cytotoxin activity, and cytotoxin-induced changes in E. coli O157:H7 colonization. Given the known lack of Stx1 activity in cattle, we examined the effects of LSPA-6 genotype, strain origin (human versus bovine), and cytotoxin activity on E. coli O157:H7 colonization of cattle.     

Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels

The regulatory mechanism of Ca2+ influx into the cytosol from the extracellular space in non-excitable cells is not clear. The "capacitative calcium entry" (CCE) hypothesis suggested that Ca2+ influx is triggered by the IP(3)-mediated emptying of the intracellular Ca2+ stores. However, there is no clear evidence for CCE and its mechanism remains elusive. In the present work, we have provided the reported evidences to show that inhibition of IP(3)-dependent Ca2+ release does not affect Ca2+ influx, and the experimental protocols used to demonstrate CCE can stimulate Ca2+ influx by means other than emptying of the Ca2+ stores. In addition, we have presented the reports showing that IP(3)-mediated Ca2+ release is linked to a Ca2+ entry from the extracellular space, which does not increase cytosolic [Ca2+] prior to Ca2+ release. Based on these and other reports, we have provided a model of Ca2+ signaling in non-excitable cells, in which IP(3)-mediated emptying of the intracellular Ca2+ store triggers entry of Ca2+ directly into the store, through a plasma membrane TRPC channel. Thus, emptying and direct refilling of the Ca2+ stores are repeated in the presence of IP(3), giving rise to the transient phase of oscillatory Ca2+ release. Direct Ca2+ entry into the store is regulated by its filling status in a negative and positive manner through a Ca2+ -binding protein and Stim1/Orai complex, respectively. The sustained phase of Ca2+ influx is triggered by diacylglycerol (DAG) through the activation of another TRPC channel, independent of Ca2+ release. The plasma membrane IP(3) receptor (IP(3)R) plays an essential role in Ca2+ influx, by interacting with the DAG-activated TRPC, without the requirement of binding to IP(3).     

Synthesis and antibacterial activity of some new 1-heteroaryl-5-amino-3H/methyl-4-phenylpyrazoles

The regioselective synthesis of 1-heteroaryl-5-amino-4-phenylpyrazoles 3a-g and 1-heteroaryl-5-amino-3-methyl-4-phenylpyrazoles 3h-n was achieved by the treatment of heteroarylhydrazines 1a-g with alpha-phenylformylacetonitrile 2a and alpha-phenylacetylacetonitrile 2b, respectively. The structures of the compounds 3 were established by the combined use of 1H and 13C NMR spectroscopy. All the fourteen compounds were tested for their in vitro antibacterial activity against three Gram-positive and two Gram-negative bacteria. Six compounds 3a, 3d, 3e, 3g, 3l, and 3n from this series were found to be equipotent or more potent than the commercial antibiotics (Linezolid and Cefroxime axetil).     

NR2B-NMDA receptor mediated modulation of the tyrosine phosphatase STEP regulates glutamate induced neuronal cell death

The present study examines the role of a neuron-specific tyrosine phosphatase (STEP, striatal-enriched tyrosine phosphatase) in excitotoxic cell death. Our findings demonstrate that p38 MAPK, a stress-activated kinase that is known to play a role in the etiology of excitotoxic cell death is a substrate of STEP. Glutamate-mediated NMDA receptor stimulation leads to rapid but transient activation of p38 MAPK, which is primarily dependent on NR2A-NMDA receptor activation. Conversely, activation of NR2B-NMDA receptors leads to dephosphorylation and subsequent activation of STEP, which in turn leads to inactivation of p38 MAPK. Thus, during transient NMDA receptor stimulation, increases in STEP activity appears to limit the duration of activation of p38 MAPK and improves neuronal survival. However, if NR2B-NMDA receptor stimulation is sustained, protective effects of STEP activation are lost, as these stimuli cause significant degradation of active STEP, leading to secondary activation of p38 MAPK. Consistent with this observation, a cell transducible TAT-STEP peptide that constitutively binds to p38 MAPK attenuated neuronal cell death caused by sustained NMDA receptor stimulation. The findings imply that the activation and levels of STEP are dependent on the duration and magnitude of NR2B-NMDA receptor stimulation and STEP serves as a modulator of NMDA receptor dependent neuronal injury, through its regulation of p38 MAPK.