Measurement of action spectra of light-activated processes

We report on a new experimental technique suitable for measurement of light-activated processes, such as fluorophore transport. The usefulness of this technique is derived from its capacity to decouple the imaging and activation processes, allowing fluorescent imaging of fluorophore transport at a convenient activation wavelength. We demonstrate the efficiency of this new technique in determination of the action spectrum of the light mediated transport of rhodamine 123 into the parasitic protozoan Giardia duodenalis. (c) 2006 Society of Photo-Optical Instrumentation Engineers

Attractant and Repellent Signaling Conformers of Sensory Rhodopsin−Transducer Complexes†

ABSTRACT: Attractant and repellent signaling conformers of the dual-signaling phototaxis receptor sensory rhodopsin I and its transducer subunit (SRI-HtrI) have recently been distinguished experimentally by the opposite connection of their retinylidene protonated Schiff bases to the outwardly located periplasmic side and inwardly located cytoplasmic side. Here we show that the pKa of the outwardly located Asp76 counterion in the outwardly connected conformer is lowered by ∼1.5 units from that of the inwardly connected conformer. The pK a difference enables quantitative determination of the relative amounts of the two conformers in wild-type cells and behavioral mutants prior to photoexcitation, comparison of their absorption spectra, and determination of their relative signaling efficiency. We have shown that the onephoton excitation of the SRI-HtrI attractant conformer causes a Schiff base connectivity switch from inwardly connected to outwardly connected states in the attractant signaling photoreaction. Conversely, a second near-UV photon drives the complex back to the inwardly connected conformer in the repellent signaling photoreaction. The results suggest a model of the color-discriminating dual-signaling mechanism in which phototaxis responses (his-kinase modulation) result from the photointerconversion of the two oppositely connected SRI-HtrI conformers by one-photon and two-photon activation. Furthermore, we find that the related repellent phototaxis SRII-HtrII receptor complex has an outwardly connecte

ОСОБЕННОСТИ АЗОТНОГО ПИТАНИЯ ЯРОВОЙ ПШЕНИЦЫ ПРИ МИНИМИЗАЦИИ ОСНОВНОЙ ОБРАБОТКИ ЧЕРНОЗЕМА ВЫЩЕЛОЧЕННОГО НОВОСИБИРСКОГО ПРИОБЬЯ

The paper aims to study the characteristics of spring wheat nitrogen nutrition in crop rotation in the field under minimum primary soil tillage. The research was carried out in the multifactor stationary field experiment in 2002–2013. The work examined the seasonal dynamics of soil nitrate nitrogen and its withdrawal incorporated in agrophytocenose in 4 variants of soil tillage systems in the fields of grain fallow rotation in the extensive background, it also studied spring wheat productivity in extensive and intensive backgrounds. It was established that under the conditions of West Siberia forest-steppe and in its black soils leached by bare black fallow with tillage, the amount of mineral nitrogen went up 2.5 and 2 times as much as the initial value over the fallow period and bare early minimum fallow period, respectively. In the spring prior to wheat sowing in the fallow, nitrogen content in a 1-meter soil layer constituted 159 kg/ha when sown in the tillage, which is 1.2 and 1.3 times as much as sown in the minimum fallow and “zero” tillage, respectively. Wheat yielding when grown in the fallow in extensive and intensive backgrounds made up 2.66–2.83 and 3.53–3.61 t/ha, respectively, and in fact, did not depend upon the techniques of the fallow preparation. In the repeated sowings, wheat productivity was 1.1–1.3 times higher in the extensive background in the tillage (1.29–1.66 t/ha) than that in the minimum tillage. With optimized mineral nutrition in plants and phytosanitary situation in seedings the productivity of wheat in field crop rotations went up 2.1–2.8 times as much without any considerable differences in soil tillage variants. Agrophytocenose fallowed was characterized by the highest indexes (125.0–130.3 kg/ha) for nitrate nitrogen withdrawn from the soil somewhat lowering from tillage to minimum cultivations. Herewith, the withdrawal of the element concerned from the soil with weeds in the extensive background built up from 8.6 kg/ha in the black fallow to 14.0 kg/ha for the early minimal fallow, for grain predecessors it went up from 15.7 in the tillage to 26.9 kg/ha in “zero’ tillage.Цель настоящей работы – изучение особенностей азотного питания яровой пшеницы в  полевом севообороте при минимизации основной обработки почвы. Исследования проводили в многофакторном стационарном полевом опыте в 2002–2013 гг. Изучали сезонную динамику со- держания нитратного азота в почве и вынос его с агрофитоценозом в четырех вариантах систем обработки почвы в  полях зернопарового севооборота на экстенсивном фоне, а  продуктивность яровой пшеницы  – на экстенсивном и  интенсивном фонах. Установлено, что в  условиях лесостепи Западной Сибири на чернозёмах выщелоченных в чистом черном пару со вспашкой общее количество минерального азота за период парования возросло в 2,5 раза, в чистом раннем минимальном пару – в 2 раза в сравнении с исходным значением. Весной перед посевом пшеницы по пару содержание азота в метровом слое почвы составило 159 кг/га по вспашке, что в 1,2 раза больше, чем по минимальной, и в 1,3 раза в сравнении с «нулевой» обработкой. Урожайность пшеницы по пару на экстенсивном и интенсивном фонах составила 2,66–2,83 и 3,53–3,61 т/га соответственно и практически не зависела от способов его подготовки. На повторных посевах урожайность пшеницы на экстенсивном фоне по вспашке (1,29–1,66 т/га) была в  1,1–1,3 раза больше, чем по минимальным обработкам. При оптимизации минерального питания растений и фитосанитарной ситуации посевов продуктивность пшеницы в полях севооборота увеличивалась в 2,1–2,8 раза без существенных различий по вариантам обработки почвы. Агрофитоценоз, идущий по пару, характеризовался наибольшими показателями (125,0–130,3  кг/га) по выносу нитратного азота из почвы, несколько снижаясь от вспашки к минимальным обработкам. При этом вынос данного элемента из почвы с сорняками на экстенсивном фоне нарастал от 8,6 кг/га по черному пару со вспашкой до 14,0 кг/га по раннему минимальному пару, по зерновым предшественникам – от 15,7 по вспашке до 26,9 кг/га по «нулевой» обработке

Light-evoked Somatosensory Perception of Transgenic Rats That Express Channelrhodopsin-2 in Dorsal Root Ganglion Cells

In vertebrate somatosensory systems, each mode of touch-pressure, temperature or pain is sensed by sensory endings of different dorsal root ganglion (DRG) neurons, which conducted to the specific cortical loci as nerve impulses. Therefore, direct electrical stimulation of the peripheral nerve endings causes an erroneous sensation to be conducted by the nerve. We have recently generated several transgenic lines of rat in which channelrhodopsin-2 (ChR2) transgene is driven by the Thy-1.2 promoter. In one of them, W-TChR2V4, some neurons were endowed with photosensitivity by the introduction of the ChR2 gene, coding an algal photoreceptor molecule. The DRG neurons expressing ChR2 were immunohistochemically identified using specific antibodies to the markers of mechanoreceptive or nociceptive neurons. Their peripheral nerve endings in the plantar skin as well as the central endings in the spinal cord were also examined. We identified that ChR2 is expressed in a certain population of large neurons in the DRG of W-TChR2V4. On the basis of their morphology and molecular markers, these neurons were classified as mechanoreceptive but not nociceptive. ChR2 was also distributed in their peripheral sensory nerve endings, some of which were closely associated with CK20-positive cells to form Merkel cell-neurite complexes or with S-100-positive cells to form structures like Meissner's corpuscles. These nerve endings are thus suggested to be involved in the sensing of touch. Each W-TChR2V4 rat showed a sensory-evoked behavior in response to blue LED flashes on the plantar skin. It is thus suggested that each rat acquired an unusual sensory modality of sensing blue light through the skin as touch-pressure. This light-evoked somatosensory perception should facilitate study of how the complex tactile sense emerges in the brain

Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin

BACKGROUND: Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages over channelrhodopsin-2 (ChR2) in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents. METHODOLOGY/PRINCIPAL FINDINGS: The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR) with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp). The ChRGR was also expressed in the motor cortical neurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5-10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs) and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5-10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex. CONCLUSIONS/SIGNIFICANCE: The opto-current-clamp study suggests that the depolarization of a small number of neurons wakes up the motor cortical network over some critical point to the activated state

Stress Sensors and Signal Transducers in Cyanobacteria

In living cells, the perception of environmental stress and the subsequent transduction of stress signals are primary events in the acclimation to changes in the environment. Some molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Based on genomic information, a systematic approach has been applied to the solution of this problem in cyanobacteria, involving mutagenesis of potential sensors and signal transducers in combination with DNA microarray analyses for the genome-wide expression of genes. Forty-five genes for the histidine kinases (Hiks), 12 genes for serine-threonine protein kinases (Spks), 42 genes for response regulators (Rres), seven genes for RNA polymerase sigma factors, and nearly 70 genes for transcription factors have been successfully inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Screening of mutant libraries by genome-wide DNA microarray analysis under various stress and non-stress conditions has allowed identification of proteins that perceive and transduce signals of environmental stress. Here we summarize recent progress in the identification of sensory and regulatory systems, including Hiks, Rres, Spks, sigma factors, transcription factors, and the role of genomic DNA supercoiling in the regulation of the responses of cyanobacterial cells to various types of stress