Optogenetic manipulation of stomatal kinetics improves carbon assimilation, water use, and growth.

Stomata serve dual and often conflicting roles, facilitating carbon dioxide influx into the plant leaf for photosynthesis and restricting water efflux via transpiration. Strategies for reducing transpiration without incurring a cost for photosynthesis must circumvent this inherent coupling of carbon dioxide and water vapor diffusion. We expressed the synthetic, light-gated K+ channel BLINK1 in guard cells surrounding stomatal pores in Arabidopsis to enhance the solute fluxes that drive stomatal aperture. BLINK1 introduced a K+ conductance and accelerated both stomatal opening under light exposure and closing after irradiation. Integrated over the growth period, BLINK1 drove a 2.2-fold increase in biomass in fluctuating light without cost in water use by the plant. Thus, we demonstrate the potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation.

Habitat, applications and genomics of the aerobic, thermophilic genus Geobacillus.

Thermophilic bacteria belonging to Bacillus genetic group 5 have been reclassified as being members of Geobacillus gen. nov., with G. stearothermophilus as the type strain. Geobacillus species, literally meaning earth or soil Bacillus, are widely distributed and readily isolated from natural and man-made thermophilic biotopes. Work within our group has however shown that an abundance of genetically distinct Geobacillus isolates can be obtained from temperate Irish soils. As with many thermophiles there is considerable interest in potential industrial application of these bacteria and their gene products. This review describes two novel applications for Geobacillus isolates, firstly in the metabolism of the herbicide glyphosate and secondly in the metabolism of quorum-sensing signal molecules from Gram-negative bacteria. Finally the current state of the art is described for Bacillus genomics, with details given of three independent genome-sequencing projects of Geobacillus isolates.

Regulation of backpropagating action potentials in mitral cell lateral dendrites by A-type potassium currents.

Dendrodendritic synapses, distributed along mitral cell lateral dendrites, provide powerful and extensive inhibition in the olfactory bulb. Activation of inhibition depends on effective penetration of action potentials into dendrites. Although action potentials backpropagate with remarkable fidelity in apical dendrites, this issue is controversial for lateral dendrites. We used paired somatic and dendritic recordings to measure action potentials in proximal dendritic segments (0-200 microm from soma) and action potential-generated calcium transients to monitor activity in distal dendritic segments (200-600 microm from soma). Somatically elicited action potentials were attenuated in proximal lateral dendrites. The attenuation was not due to impaired access resistance in dendrites or to basal synaptic activity. However, a single somatically elicited action potential was sufficient to evoke a calcium transient throughout the lateral dendrite, suggesting that action potentials reach distal dendritic compartments. Block of A-type potassium channels (I(A)) with 4-aminopyridine (10 mM) prevented action potential attenuation in direct recordings and significantly increased dendritic calcium transients, particularly in distal dendritic compartments. Our results suggest that I(A) may regulate inhibition in the olfactory bulb by controlling action potential amplitudes in lateral dendrites.

Quantitative and functional differences in CD8+ lymphocyte responses in resolved acute and chronic hepatitis C virus infection.

CD8+ T lymphocyte responses are important in the clearance of viral infections. In chronic infections they may contribute to pathogenesis. To investigate the role of CD8+ T lymphocyte responses in viral clearance and chronic hepatitis C we have compared hepatitis C virus (HCV) specific cytotoxicity and interferon-gamma (IFN-gamma) production in patients with resolved-acute, and chronic HCV infection. CD8+ T cell responses to a panel of 13 HCV T cell peptide epitopes were studied using Elispot assays of IFN-gamma production and chromium release cytotoxicity assays. Responses of seven patients with resolved acute HCV infection were compared with those of 14 chronically infected patients. HCV-specific cytotoxicity differentiated the two populations of patients. The majority (71%) of patients with resolved acute infection tested positive to 42% of relevant peptides compared with the minority (28%) of patients with chronic hepatitis C (P=0.03) who responded to only 8% of relevant peptides (P=0.0009). In contrast, HCV-specific IFN-gamma production was detected in 86% of patients with either resolved or chronic infection in response to 42% and 35%, respectively, of relevant peptides tested (not significant). In patients with chronic infection the magnitude of the HCV-specific IFN-gamma production was inversely correlated to viral load (R2=0.52; P=0.042). Failure to clear HCV infection may be attributable to the presence of noncytolytic IFN-gamma producing CD8+ T lymphocytes in chronically infected patients. However these CD8+ T cells may play a beneficial role in contributing to the control of viral load in chronic hepatitis C.

The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin.

The plant blue light receptor, phot1, a member of the phototropin family, is a plasma membrane-associated flavoprotein that contains two ( approximately 110 amino acids) flavin-binding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. E. coli-expressed LOV domains bind riboflavin-5'-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Blue light excitation of the LOV2 domain generates, in less than 30 ns, a transient approximately 660 nm-absorbing species that spectroscopically resembles a flavin triplet state. This putative triplet state subsequently decays with a 4-micros time constant into a 390 nm-absorbing metastable form. The LOV2 domain (450 nm) recovers spontaneously with half-times of approximately 50 s. It has been shown that the metastable species is likely a flavin-cysteine (Cys(39) thiol) adduct at the flavin C(4a) position. A LOV2C39A mutant generates the early photoproduct but not the adduct. Titrations of LOV2 using chromophore fluorescence as an indicator suggest that Cys(39) exists as a thiolate.

Five-year follow-up of patients with primary antibody deficiencies following an outbreak of acute hepatitis C.

This report is the 5-year follow-up of those 25 UK patients with primary antibody deficiencies infected with hepatitis C virus (HCV), type 1a, from one batch of contaminated anti-HCV-screened intravenous immunoglobulin in 1993-1994. Of these patients, who were reported previously (1, 2), 2 cleared HCV spontaneously, 18 received early interferon-alpha (IFN) treatment for 6 months, and 5 declined treatment or treatment was contraindicated. The clinical course of this cohort was followed prospectively using serial standardized questionnaires. Seven patients (54% of those who had completed therapy) had a sustained response (normal transaminase levels, negative serum HCV RNA) for 5 years posttreatment. Eight patients died: 3 from decompensated cirrhosis, 2 from pneumonia but with evidence of liver failure, and 3 from unrelated causes. One further patient developed decompensated cirrhosis but was successfully transplanted. Seven patients remain chronically infected; only 1 patient is symptomatic but 1 further patient has evidence of progressive fibrosis on liver histology. In conclusion, within 5 years, rapid end-stage HCV liver disease has been seen in 6/25 (24%) patients. Seven patients, (54% of those fully treated) remain well after treatment, making 9/25 (36% of the cohort) clear of virus after 5 years. Those who completed early treatment with IFN had a relatively high sustained response rate compared to previous studies in both immunodeficient and immunocompetent patients.

Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation.

UV-A/blue light acts to regulate a number of physiological processes in higher plants. These include light-driven chloroplast movement and phototropism. The NPH1 gene of Arabidopsis encodes an autophosphorylating protein kinase that functions as a photoreceptor for phototropism in response to low-intensity blue light. However, nph1 mutants have been reported to exhibit normal phototropic curvature under high-intensity blue light, indicating the presence of an additional phototropic receptor. A likely candidate is the nph1 homologue, npl1, which has recently been shown to mediate the avoidance response of chloroplasts to high-intensity blue light in Arabidopsis. Here we demonstrate that npl1, like nph1, noncovalently binds the chromophore flavin mononucleotide (FMN) within two specialized PAS domains, termed LOV domains. Furthermore, when expressed in insect cells, npl1, like nph1, undergoes light-dependent autophosphorylation, indicating that npl1 also functions as a light receptor kinase. Consistent with this conclusion, we show that a nph1 npl1 double mutant exhibits an impaired phototropic response under both low- and high-intensity blue light. Hence, npl1 functions as a second phototropic receptor under high fluence rate conditions and is, in part, functionally redundant to nph1. We also demonstrate that both chloroplast accumulation in response to low-intensity light and chloroplast avoidance movement in response to high-intensity light are lacking in the nph1 npl1 double mutant. Our findings therefore indicate that nph1 and npl1 show partially overlapping functions in two different responses, phototropism and chloroplast relocation, in a fluence rate-dependent manner.

Tufted cell dendrodendritic inhibition in the olfactory bulb is dependent on NMDA receptor activity.

Mitral and tufted cells constitute the primary output cells of the olfactory bulb. While tufted cells are often considered as "displaced" mitral cells, their actual role in olfactory bulb processing has been little explored. We examined dendrodendritic inhibition between tufted cells and interneurons using whole cell voltage-clamp recording. Dendrodendritic inhibitory postsynaptic currents (IPSCs) generated by depolarizing voltage steps in tufted cells were completely blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2amino-5-phosphonopentanoic acid (D,L-AP5), whereas the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 2-3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f] quinoxaline-7-sulfonamide (NBQX) had no effect. Tufted cells in the external plexiform layer (EPL) and in the periglomerular region (PGR) showed similar behavior. These results indicate that NMDA receptor-mediated excitation of interneurons drives inhibition of tufted cells at dendrodendritic synapses as it does in mitral cells. However, the spatial extent of lateral inhibition in tufted cells was much more limited than in mitral cells. We suggest that the sphere of influence of tufted cells, while qualitatively similar to mitral cells, is centered on only one or a few glomeruli.

Phototropins: a new family of flavin-binding blue light receptors in plants.

Phototropin is the designation originally assigned to a recently characterized chromoprotein that serves as a photoreceptor for phototropism. Phototropin is a light-activated autophosphorylating serine/threonine kinase that binds two flavin mononucleotide (FMN) molecules that function as blue light-absorbing chromophores. Each FMN molecule is bound in a rigid binding pocket within specialized PAS (PER-ARNT-SIM superfamily) domains, known as LOV (light, oxygen, or voltage) domains. This article reviews the detailed photobiological and biochemical characterization of the light-activated phosphorylation reaction of phototropin and follows the sequence of events leading to the cloning, sequencing, and characterization of the gene and the subsequent biochemical characterization of its encoded protein. It then considers recent biochemical and photochemical evidence that light activation of phototropin involves the formation of a cysteinyl adduct at the C(4a) position of the FMN chromophores. Adduct formation causes a major conformational change in the chromophores and a possible conformational change in the protein moiety as well. The review concludes with a brief discussion of the evidence for a second phototropin-like protein in Arabidopsis and rice. Possible roles for this photoreceptor are discussed.

Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin.

The plant photoreceptor phototropin is an autophosphorylating serine-threonine protein kinase activated by UV-A/blue light. Two domains, LOV1 and LOV2, members of the PAS domain superfamily, mediate light sensing by phototropin. Heterologous expression studies have shown that both domains function as FMN-binding sites. Although three plant blue light photoreceptors, cry1, cry2, and phototropin, have been identified to date, the photochemical reactions underlying photoactivation of these light sensors have not been described so far. Herein, we demonstrate that the LOV domains of Avena sativa phototropin undergo a self-contained photocycle characterized by a loss of blue light absorbance in response to light and a spontaneous recovery of the blue light-absorbing form in the dark. Rate constants and quantum efficiencies for the photoreactions indicate that LOV1 exhibits a lower photosensitivity than LOV2. The spectral properties of the photoproduct produced for both LOV domains are unrelated to those found for photoreduced flavins and flavoproteins, but are consistent with those of a flavin-cysteinyl adduct. Flavin-thiol adducts are generally short-lifetime reaction intermediates formed during the flavoprotein-catalyzed reduction of protein disulfides. By site-directed mutagenesis, we have identified several amino acid residues within the putative chromophore binding site of LOV1 and LOV2 that appear to be important for FMN binding and/or the photochemical reactivity. Among those is Cys39, which plays an important role in the photochemical reaction of the LOV domains. Replacement of Cys39 with Ala abolished the photochemical reactions of both LOV domains. We therefore propose that light sensing by the phototropin LOV domains occurs via the formation of a stable adduct between the FMN chromophore and Cys39.

Native N-methyl-D-aspartate receptors containing NR2A and NR2B subunits have pharmacologically distinct competitive antagonist binding sites.

The pharmacological properties of native N-methyl-D-aspartate (NMDA) receptors were determined in rat brain sections with quantitative autoradiography of [(3)H](E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) binding. With five competitive antagonists as displacers, two subpopulations of binding sites were observed in the horizontal plane of section examined. These two populations corresponded anatomically to NR2A and NR2B subunits. Quantitative analysis of NR2A-like and NR2B-like binding sites was enabled by examining the cerebellar granule cell layer, which expresses NR2A and NR2C subunits, and the medial striatum, which predominately expresses NR2B subunits. The antagonists (R)-(E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid and (R)-2-amino-5-phosphonopentanoate (D-AP5) displayed similar affinities at cerebellar NMDA receptors and medial striatal NMDA receptors. In contrast, the NMDA receptor antagonists (+/-)-6-(1H-Tetrazol-5-ylmethyl)decahydroisoquinoline- 3-carboxylic acid, (S)-alpha-amino-5-(phosphonomethyl)[1,1'-biphenyl]-3-propanoic acid, and (+/-)-cis-4-(4-phenylbenzoyl) piperazine-2,3-dicarboxylic acid displayed varied, higher affinities at medial striatal NMDA receptors than at cerebellar NMDA receptors. For the five antagonists, there was a strong correlation (r = 0.9) between the cerebellar K(i)/medial striatum K(i) ratio and the NR2A K(i)/NR2B K(i) ratio for recombinant receptors. Thus, [(3)H]CGP39653 labels two pharmacologically distinct populations of NMDA receptors that have pharmacological and anatomical properties consistent with NR2A and NR2B subunits. Because native NR2A- and NR2B-containing receptors are pharmacologically distinct, it should be possible to develop NR2A- and NR2B-selective glutamate site antagonists.

Immune selection and genetic sequence variation in core and envelope regions of hepatitis C virus.

How Hepatitis C Virus (HCV) causes persistent infection is unknown. One hypothesis is that HCV evades the host immune response through mutation in immune epitopes. We have investigated mutations in the HCV genome to see if they cluster within immune epitopes; and we have studied the effect of antibody deficiency on mutation rates. We studied patients with chronic hepatitis C, 3 with antibody deficiency and 3 with normal immunity. Regions of the core and envelope genes of HCV, encoding cytotoxic (CTL), and B cell epitopes were sequenced at 2 time points, 2 years apart. The diversity of quasispecies increased with time. The HCV genetic mutation rate was higher than previously predicted. The cryptic nucleotide mutation rate in core was similar to that observed in envelope, suggesting that the error rate of the HCV RNA polymerase is similar in both regions. In contrast, the coding mutation rate was decreased in core and increased in envelope. No genetic mutation was seen in any of the core CTL epitopes despite detectable cellular responses. All patients had mutations within a previously described envelope CTL epitope but did not exhibit immune responses to either index or mutated peptides. There was no difference in mutation rates in any cellular or humoral epitopes between patients with antibody deficiency and normal immunity. Thus we have found no evidence that mutations were selected by T-lymphocytes or antibodies. These findings implicate alternative virus-host interactions in the selection of HCV mutations.

LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide.

Phototropism, the bending response of plant organs to or away from a directional light source, is one of the best studied blue light responses in plants. Although phototropism has been studied for more than a century, recent advances have improved our understanding of the underlying signaling mechanisms involved. The NPH1 gene of Arabidopsis thaliana encodes a blue light-dependent autophosphorylating protein kinase with the properties of a photoreceptor for phototropism. NPH1 apoprotein noncovalently binds FMN to form the holoprotein nph1. The N-terminal region of the protein contains two LOV (light, oxygen, or voltage) domains that share homology with sensor proteins from a diverse group of organisms. These include the bacterial proteins NIFL and AER, both of which bind FAD, and the phy3 photoreceptor from Adiantium capillus-veneris. The LOV domain has therefore been proposed to reflect a flavin-binding site, regulating nph1 kinase activity in response to blue light-induced redox changes. Herein we demonstrate that the LOV domains of two nph1 proteins and phy3 bind stoichiometric amounts of FMN when expressed in Escherichia coli. The spectral properties of the chromopeptides are similar to the action spectrum for phototropism, implying that the LOV domain binds FMN to function as a light sensor. Thus, our findings support the earlier model that nph1 is a dual-chromophoric flavoprotein photoreceptor regulating phototropic responses in higher plants. We therefore propose the name phototropin to designate the nph1 holoprotein.

Combination therapy for chronic hepatitis C: interferon and ribavirin.

Hepatitis C virus (HCV) infection is one of the commonest causes of liver cirrhosis and hepatocellular carcinoma. This review deals with treatment of chronic HCV infection with a combination of interferon and ribavirin. Recent trials have shown that approximately 40% of patients will clear HCV with combination treatment. This is an important advance in the treatment of this serious viral infection.

Arabidopsis contains at least four independent blue-light-activated signal transduction pathways.

We have investigated the stomatal and phototropic responses to blue light of a number of single and double mutants at various loci that encode proteins involved in blue-light responses in Arabidopsis. The stomatal responses of light-grown mutant plants (cry1, cry2, nph1, nph3, nph4, cry1cry2, and nph1cry1) did not differ significantly from those of their wild-type counterparts. Second positive phototropic responses of etiolated mutant seedlings, cry1, cry2, cry1cry2, and npq1-2, were also similar to those of their wild-type counterparts. Although npq1 and single and double cry1cry2 mutants showed somewhat reduced amplitude for first positive phototropism, threshold, peak, and saturation fluence values for first positive phototropic responses of etiolated seedlings did not differ from those of wild-type seedlings. Similar to the cry1cry2 double mutants and to npq1-2, a phyAphyB mutant showed reduced curvature but no change in the position or shape of the fluence-response curve. By contrast, the phototropism mutant nph1-5 failed to show phototropic curvature under any of the irradiation conditions used in the present study. We conclude that the chromoproteins cry1, cry2, nph1, and the blue-light photoreceptor for the stomatal response are genetically separable. Moreover, these photoreceptors appear to activate separate signal transduction pathways.

Serum levels of total and free IGF-I and IGFBP-3 are increased and maintained in long-term training.

The goals of this study were to determine whether the long-term training regimens experienced by competitive collegiate swimmers would result in altered levels of total and free serum insulin-like growth factor I (IGF-I) as well as IGF-binding proteins (BP) IGFBP-1 and -3. Two male (Teams 1M and 2M) and one female (Team 2F) teams were studied at the start of training, after 2 mo of training, after 4 mo (2-4 mo had the highest volume of training), after 5 mo (near the end of tapering; only for Team 1M), and several days after training was over. For Team 1M, total IGF-I concentrations were increased by 76% after 4 mo and were subsequently maintained at this level. Total IGF-I responses were more variable for Teams 2F and 2M. Free IGF-I levels were increased nearly twofold for all teams at 2 mo and were maintained or increased further with subsequent training. Only the levels of free IGF-I for Team 1M returned to pretraining values after training had ended. Training had little effect on IGFBP-1 levels. For all teams, serum IGFBP-3 was elevated by 4 mo of training (for Team 2F it was increased at 2 mo) by 30-97% and remained at these higher levels thereafter. The ratio of total IGF-I to IGFBP-3 was not increased by training in any group. These data indicate that serum levels of total and free IGF-I and total IGFBP-3 can be increased with intense training and maintained with reduced training (tapering). The findings show that changes in free IGF-I levels are not accounted for by alterations in the total IGF-I/IGFBP-3 complex or in IGFBP-3 levels and indicate that there are other important determinants of free IGF-I.

Insulin causes a transient tyrosine phosphorylation of NR2A and NR2B NMDA receptor subunits in rat hippocampus.

NMDA receptors play a critical role in various aspects of CNS function. Hence, it is important to identify mechanisms that regulate NMDA receptor activity. We have shown previously that insulin rapidly potentiates NMDA receptor activity in both native and recombinant expression systems. Here we report that insulin causes a transient phosphorylation of NR2A and NR2B NMDA receptor subunits on tyrosine residues. Rat hippocampal slices were exposed to 1 microM insulin for 20 and 60 min and then solubilized. NR2A and NR2B subunits were immunoprecipitated and probed for tyrosine phosphorylation. Insulin incubation of hippocampal slices for 20 min elicited an increase in tyrosine phosphorylation to 176 +/- 16% (NR2A) and 203 +/- 15% (NR2B) of control levels. In contrast, 60 min of insulin incubation did not alter NR2 tyrosine phosphorylation levels (NR2A: 85 +/- 13% of control; NR2B: 93 +/- 10% of control). Although the consequence of insulin-stimulated tyrosine phosphorylation is unknown, it is possible that this site(s) is responsible for insulin potentiation of NMDA receptor activity. This possibility is consistent with our earlier finding that insulin potentiates hippocampal NMDA receptor activity after 20 min, but not after 60 min, of insulin exposure.

Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism.

The NPH1 gene of Arabidopsis thaliana encodes a 120-kilodalton serine-threonine protein kinase hypothesized to function as a photoreceptor for phototropism. When expressed in insect cells, the NPH1 protein is phosphorylated in response to blue light irradiation. The biochemical and photochemical properties of the photosensitive protein reflect those of the native protein in microsomal membranes. Recombinant NPH1 noncovalently binds flavin mononucleotide, a likely chromophore for light-dependent autophosphorylation. The fluorescence excitation spectrum of the recombinant protein is similar to the action spectrum for phototropism, consistent with the conclusion that NPH1 is an autophosphorylating flavoprotein photoreceptor mediating phototropic responses in higher plants.