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1.
Commun Biol ; 4(1): 362, 2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33742139

RESUMO

Microbial rhodopsins are photoreceptive membrane proteins, which are used as molecular tools in optogenetics. Here, a machine learning (ML)-based experimental design method is introduced for screening rhodopsins that are likely to be red-shifted from representative rhodopsins in the same subfamily. Among 3,022 ion-pumping rhodopsins that were suggested by a protein BLAST search in several protein databases, the ML-based method selected 65 candidate rhodopsins. The wavelengths of 39 of them were able to be experimentally determined by expressing proteins with the Escherichia coli system, and 32 (82%, p = 7.025 × 10-5) actually showed red-shift gains. In addition, four showed red-shift gains >20 nm, and two were found to have desirable ion-transporting properties, indicating that they would be potentially useful in optogenetics. These findings suggest that data-driven ML-based approaches play effective roles in the experimental design of rhodopsin and other photobiological studies. (141/150 words).

2.
Elife ; 92020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33103999

RESUMO

Diverse mechanosensory neurons detect different mechanical forces that can impact animal behavior. Yet our understanding of the anatomical and physiological diversity of these neurons and the behaviors that they influence is limited. We previously discovered that grooming of the Drosophila melanogaster antennae is elicited by an antennal mechanosensory chordotonal organ, the Johnston's organ (JO) (Hampel et al., 2015). Here, we describe anatomically and physiologically distinct JO mechanosensory neuron subpopulations that each elicit antennal grooming. We show that the subpopulations project to different, discrete zones in the brain and differ in their responses to mechanical stimulation of the antennae. Although activation of each subpopulation elicits antennal grooming, distinct subpopulations also elicit the additional behaviors of wing flapping or backward locomotion. Our results provide a comprehensive description of the diversity of mechanosensory neurons in the JO, and reveal that distinct JO subpopulations can elicit both common and distinct behavioral responses.

3.
Sci Rep ; 10(1): 15092, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934309

RESUMO

This study quantitatively assessed the population-wide lead poisoning conditions in Kabwe, Zambia, a town with severe lead pollution. While existing data have reported concerning blood lead levels (BLLs) of residents in pollution hotspots, the data representing the entire population are lacking. Further, selection bias is a concern. Given the lack of compulsory testing schemes, BLLs have been observed from voluntary participants in blood sampling surveys, but such data can represent higher or lower BLLs than the population average because of factors simultaneously affecting participation and BLLs. To illustrate the lead poisoning conditions of the population, we expanded the focus of our surveys and then econometrically estimated the BLLs of individuals representing the population, including those not participating in blood sampling, using background geographic, demographic, and socioeconomic information. The estimated population mean BLL was 11.9 µg/dL (11.6-12.1, 95% CI), lower than existing data because of our wide focus and correction of selection bias. However, the scale of lead poisoning remained immense and 74.9% of residents had BLLs greater than 5 µg/dL, the standard reference level for lead poisoning. Our estimates provide a deeper understanding of the problem and a foundation for policy intervention designs.

4.
Photochem Photobiol Sci ; 19(10): 1326-1331, 2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-32935701

RESUMO

Cryptochromes (CRYs) are blue-light receptors involved in photomorphogenesis in plants. Flavin adenine dinucleotide (FAD) is one of the chromophores of cryptochromes; its resting state oxidized form is converted into a signalling state neutral semiquionod radical (FADH˙) form. Studies have shown that cryptochrome 1 from Arabidopsis thaliana (AtCRY1) can bind ATP at its photolyase homology region (PHR), resulting in accumulation of FADH˙ form. This study used light-induced difference Fourier transform infrared spectroscopy to investigate how ATP influences structural changes in AtCRY1-PHR during the photoreaction. In the presence of ATP, there were large changes in the signals from the protein backbone compared with in the absence of ATP. The deprotonation of a carboxylic acid was observed only in the presence of ATP; this was assigned as aspartic acid (Asp) 396 through measurement of Asp to glutamic acid mutants. This corresponds to the protonation state of Asp396 estimated from the reported pKa values of Asp396; that is, the side chain of Asp396 is deprotonated and protonated for the ATP-free and -bound forms, respectively, in our experimental condition at pH8. Therefore, Asp396 acts a proton donor to FAD when it is ptotonated. It was indicated that the protonation/deprotination process of Asp396 is correlated with the accunumulation of FADH˙ and protein conformational changes.

5.
Chemosphere ; 243: 125412, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31995873

RESUMO

Childhood lead (Pb) poisoning has devastating effects on neurodevelopment and causes overt clinical signs including convulsions and coma. Health effects including hypertension and various reproductive problems have been reported in adults. Historical Pb mining in Zambia's Kabwe town left a legacy of environmental pollution and childhood Pb poisoning. The current study aimed at establishing the extent of Pb poisoning and exposure differences among family members in Kabwe as well as determining populations at risk and identify children eligible for chelation therapy. Blood samples were collected in July and August 2017 from 1190 household members and Pb was measured using a portable LeadCare-II analyser. Participants included 291 younger children (3-months to 3-years-old), 271 older children (4-9-years-old), 412 mothers and 216 fathers from 13 townships with diverse levels of Pb contamination. The Blood Lead Levels (BLL) ranged from 1.65 to 162  µg/dL, with residents from Kasanda (mean 45.7  µg/dL) recording the highest BLL while Hamududu residents recorded the lowest (mean 3.3  µg/dL). Of the total number of children sampled (n = 562), 23% exceeded the 45  µg/dL, the threshold required for chelation therapy. A few children (5) exceeded the 100  µg/dL whereas none of the parents exceeded the 100  µg/dL value. Children had higher BLL than parents, with peak BLL-recorded at the age of 2-years-old. Lead exposure differences in Kabwe were attributed to distance and direction from the mine, with younger children at highest risk. Exposure levels in parents were equally alarming. For prompt diagnosis and treatment, a portable point-of-care devise such as a LeadCare-II would be preferable in Kabwe.


Assuntos
Exposição Ambiental/análise , Intoxicação por Chumbo/epidemiologia , Chumbo/sangue , Chumbo/toxicidade , Adulto , Criança , Pré-Escolar , Poluição Ambiental/análise , Pai , Feminino , Humanos , Lactente , Masculino , Mineração , Mães , Registros , Reprodução , Manejo de Espécimes , Zâmbia/epidemiologia
6.
Brain Nerve ; 71(6): 599-609, 2019 Jun.
Artigo em Japonês | MEDLINE | ID: mdl-31171757

RESUMO

Many animals use acoustic information to recognize conspecifics. The time interval between acoustic elements is a property that characterizes a particular sound. In vertebrates and invertebrates, a specific time interval between acoustic elements is represented as a selective pattern of neuronal activity. Excitatory and inhibitory inputs to the auditory neural circuit generate this selectivity. However, the direct causal link between these inhibitory systems and the behavioral response of an animal to a sound with a specific time interval remains unknown. To tackle this question, in this study, we used Drosophila melanogaster, which has a courtship song with a species-specific time interval. Song information in these flies is transmitted along the main songrelay neural pathway, through which the time interval selectivity of the neurons is sequentially transformed. Herein, we examined the mechanism that shapes the selectivity of the key secondary auditory neurons in this pathway, AMMC-B1. Calcium imaging experiments suggested that AMMC-B1 neurons receive GABAergic inhibitory inputs. Anatomical analysis suggested that two GABAergic neurons configure the feed-forward pathways onto the excitatory pathway between AMMC-B1 neurons and their upstream neurons. Calcium imaging and behavioral analysis suggested that each GABAergic neuron shaped the response selectivity of AMMC-B1 neurons, and suppressed the songresponse behavior of the flies. Based on these results, it therefore appears that GABAergic inhibitory feed-forward pathways shape the tuning pattern of AMMC-B1 neurons and adjust the fly's behavioral response to the song.


Assuntos
Corte , Drosophila melanogaster/fisiologia , Neurônios GABAérgicos/fisiologia , Vias Neurais , Vocalização Animal , Animais , Som
7.
Photochem Photobiol ; 95(5): 1116-1121, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31066906

RESUMO

Definition of rhodopsin is the retinal-binding membrane protein with the Schiff base linkage at a lysine on the 7th transmembrane helix. However, ~ 600 microbial rhodopsins lack retinal-binding lysine at the corresponding position (Rh-noK) among ~ 5500 known microbial rhodopsins, suggesting that Rh-noK has each functional role without chromophore. Here, we report successful functional recovery of Rh-noK. Two Rh-noKs from bacteria were heterologously expressed in Escherichia coli, which exhibited no color. When retinal-binding lysine was introduced, one of them gained visible color. Additional mutation of the Schiff base counterion further gained proton-pumping activity. Successful engineered functional recovery such as visible color and proton-pump activity suggests that the Rh-noK protein forms a characteristic structure of microbial rhodopsins.


Assuntos
Lisina/metabolismo , Engenharia de Proteínas , Retinaldeído/metabolismo , Rodopsina/metabolismo , Escherichia coli/genética , Ligação Proteica
8.
J Biol Chem ; 294(10): 3432-3443, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622140

RESUMO

The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an "M" intermediate with a deprotonated Schiff base, and its recovery is much faster than that of the enzyme domain. To gain structural and mechanistic insights into the Rh domain, here we expressed and purified the transmembrane domain of Rh-PDE, Rh-PDE(TMD), and analyzed it with transient absorption, light-induced difference UV-visible, and FTIR spectroscopy methods. These analyses revealed that the "K" intermediate forms within 0.005 ms and converts into the M intermediate with a time constant of 4 ms, with the latter returning to the original state within 4 s. FTIR spectroscopy revealed that all-trans to 13-cis photoisomerization occurs as the primary event during which chromophore distortion is located at the middle of the polyene chain, allowing the Schiff base to form a stronger hydrogen bond. We also noted that the peptide backbone of the α-helix becomes deformed upon M intermediate formation. Results from site-directed mutagenesis suggested that Glu-164 is protonated and that Asp-292 acts as the only Schiff base counterion in Rh-PDE. A strong reduction of enzymatic activity in a D292N variant, but not in an E164Q variant, indicated an important catalytic role of the negative charge at Asp-292. Our findings provide further mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.


Assuntos
Membrana Celular/enzimologia , Coanoflagelados/enzimologia , Diester Fosfórico Hidrolases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Mutação , Domínios Proteicos , Rodopsina/genética , Análise Espectral
9.
J Neurosci ; 38(18): 4329-4347, 2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29691331

RESUMO

Many animals use acoustic signals to attract a potential mating partner. In fruit flies (Drosophila melanogaster), the courtship pulse song has a species-specific interpulse interval (IPI) that activates mating. Although a series of auditory neurons in the fly brain exhibit different tuning patterns to IPIs, it is unclear how the response of each neuron is tuned. Here, we studied the neural circuitry regulating the activity of antennal mechanosensory and motor center (AMMC)-B1 neurons, key secondary auditory neurons in the excitatory neural pathway that relay song information. By performing Ca2+ imaging in female flies, we found that the IPI selectivity observed in AMMC-B1 neurons differs from that of upstream auditory sensory neurons [Johnston's organ (JO)-B]. Selective knock-down of a GABAA receptor subunit in AMMC-B1 neurons increased their response to short IPIs, suggesting that GABA suppresses AMMC-B1 activity at these IPIs. Connection mapping identified two GABAergic local interneurons that synapse with AMMC-B1 and JO-B. Ca2+ imaging combined with neuronal silencing revealed that these local interneurons, AMMC-LN and AMMC-B2, shape the response pattern of AMMC-B1 neurons at a 15 ms IPI. Neuronal silencing studies further suggested that both GABAergic local interneurons suppress the behavioral response to artificial pulse songs in flies, particularly those with a 15 ms IPI. Altogether, we identified a circuit containing two GABAergic local interneurons that affects the temporal tuning of AMMC-B1 neurons in the song relay pathway and the behavioral response to the courtship song. Our findings suggest that feedforward inhibitory pathways adjust the behavioral response to courtship pulse songs in female flies.SIGNIFICANCE STATEMENT To understand how the brain detects time intervals between sound elements, we studied the neural pathway that relays species-specific courtship song information in female Drosophila melanogaster We demonstrate that the signal transmission from auditory sensory neurons to key secondary auditory neurons antennal mechanosensory and motor center (AMMC)-B1 is the first-step to generate time interval selectivity of neurons in the song relay pathway. Two GABAergic local interneurons are suggested to shape the interval selectivity of AMMC-B1 neurons by receiving auditory inputs and in turn providing feedforward inhibition onto AMMC-B1 neurons. Furthermore, these GABAergic local interneurons suppress the song response behavior in an interval-dependent manner. Our results provide new insights into the neural circuit basis to adjust neuronal and behavioral responses to a species-specific communication sound.


Assuntos
Drosophila melanogaster/fisiologia , Interneurônios/fisiologia , Comportamento Sexual Animal/fisiologia , Vocalização Animal/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Antenas de Artrópodes/fisiologia , Sinalização do Cálcio , Copulação , Feminino , Mecanorreceptores/fisiologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Receptores de GABA-A/fisiologia
10.
J Struct Funct Genomics ; 17(4): 69-81, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28012137

RESUMO

Life science research now heavily relies on all sorts of databases for genome sequences, transcription, protein three-dimensional (3D) structures, protein-protein interactions, phenotypes and so forth. The knowledge accumulated by all the omics research is so vast that a computer-aided search of data is now a prerequisite for starting a new study. In addition, a combinatory search throughout these databases has a chance to extract new ideas and new hypotheses that can be examined by wet-lab experiments. By virtually integrating the related databases on the Internet, we have built a new web application that facilitates life science researchers for retrieving experts' knowledge stored in the databases and for building a new hypothesis of the research target. This web application, named VaProS, puts stress on the interconnection between the functional information of genome sequences and protein 3D structures, such as structural effect of the gene mutation. In this manuscript, we present the notion of VaProS, the databases and tools that can be accessed without any knowledge of database locations and data formats, and the power of search exemplified in quest of the molecular mechanisms of lysosomal storage disease. VaProS can be freely accessed at http://p4d-info.nig.ac.jp/vapros/ .


Assuntos
Biologia Computacional , Bases de Dados Genéticas , Genoma , Internet , Software , Animais , Humanos , Camundongos , Conformação Proteica , Ratos , Análise de Sequência de DNA
11.
Biochemistry ; 55(30): 4173-83, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27431478

RESUMO

Ultraviolet (UV) light from the sun damages DNA by forming a cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) PP]. Photolyase (PHR) enzymes utilize near-UV/blue light for DNA repair, which is initiated by light-induced electron transfer from the fully reduced flavin adenine dinucleotide chromophore. Despite similar structures and repair mechanisms, the functions of PHR are highly selective; CPD PHR repairs CPD, but not (6-4) PP, and vice versa. In this study, we attempted functional conversion between CPD and (6-4) PHRs. We found that a triple mutant of (6-4) PHR is able to repair the CPD photoproduct, though the repair efficiency is 1 order of magnitude lower than that of wild-type CPD PHR. Difference Fourier transform infrared spectra for repair demonstrate the lack of secondary structural alteration in the mutant, suggesting that the triple mutant gains substrate binding ability while it does not gain the optimized conformational changes from light-induced electron transfer to the release of the repaired DNA. Interestingly, the (6-4) photoproduct is not repaired by the reverse mutation of CPD PHR, and eight additional mutations (total of 11 mutations) introduced into CPD PHR are not sufficient. The observed asymmetric functional conversion is interpreted in terms of a more complex repair mechanism for (6-4) repair, which was supported by quantum chemical/molecular mechanical calculation. These results suggest that CPD PHR may represent an evolutionary origin for photolyase family proteins.


Assuntos
Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Dímeros de Pirimidina/metabolismo , Substituição de Aminoácidos , Animais , Domínio Catalítico/genética , Cristalografia por Raios X , Dano ao DNA , Reparo do DNA , Desoxirribodipirimidina Fotoliase/química , Transporte de Elétrons , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Dímeros de Pirimidina/química , Dímeros de Pirimidina/efeitos da radiação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Raios Ultravioleta , Xenopus laevis
12.
Biochemistry ; 55(4): 715-23, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26719910

RESUMO

Photolyases (PHRs) repair the UV-induced photoproducts, cyclobutane pyrimidine dimer (CPD) or pyrimidine-pyrimidone (6-4) photoproduct [(6-4) PP], restoring normal bases to maintain genetic integrity. CPD and (6-4) PP are repaired by substrate-specific PHRs, CPD PHR and (6-4) PHR, respectively. Flavin adenine dinucleotide (FAD) is the chromophore of both PHRs, and the resting oxidized form (FAD(ox)), at least under in vitro purified conditions, is first photoconverted to the neutral semiquinoid radical (FADH(•)) form, followed by photoconversion into the enzymatically active fully reduced (FADH(-)) form. Previously, we reported light-induced difference Fourier transform infrared (FTIR) spectra corresponding to the photoactivation process of Xenopus (6-4) PHR. Spectral differences between the absence and presence of (6-4) PP were observed in the photoactivation process. To identify the FTIR signals where these differences appeared, we compared the FTIR spectra of photoactivation (i) in the presence and absence of (6-4) PP, (ii) of (13)C labeling, (15)N labeling, and [(14)N]His/(15)N labeling, and (iii) of H354A and H358A mutants. We successfully assigned the vibrational bands for (6-4) PP, the α-helix and neutral His residue(s). In particular, we assigned three bands to the C ═ O groups of (6-4) PP in the three different redox states of FAD. Furthermore, the changed hydrogen bonding environments of C ═ O groups of (6-4) PP suggested restructuring of the binding pocket of the DNA lesion in the process of photoactivation.


Assuntos
Desoxirribodipirimidina Fotoliase/química , Flavina-Adenina Dinucleotídeo/química , Dímeros de Pirimidina/química , Proteínas de Xenopus/química , Substituição de Aminoácidos , Animais , Domínio Catalítico , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Flavina-Adenina Dinucleotídeo/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Mutação de Sentido Incorreto , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/imunologia , Xenopus laevis
13.
Biophys Physicobiol ; 12: 139-44, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-27493863

RESUMO

Photolyases (PHRs) are DNA repair enzymes that revert UV-induced photoproducts, either cyclobutane pyrimidine dimers (CPD) or (6-4) photoproducts (PPs), into normal bases to maintain genetic integrity. (6-4) PHR must catalyze not only covalent bond cleavage, but also hydroxyl or amino group transfer, yielding a more complex mechanism than that postulated for CPD PHR. Previous mutation analysis revealed the importance of two histidines in the active center, H354 and H358 for Xenopus (6-4) PHR, whose mutations significantly lowered the enzymatic activity. Based upon highly sensitive FTIR analysis of the repair function, here we report that both H354A and H358A mutants of Xenopus (6-4) PHR still maintain their repair activity, although the efficiency is much lower than that of the wild type. Similar difference FTIR spectra between the wild type and mutant proteins suggest a common mechanism of repair in which (6-4) PP binds to the active center of each mutant, and is released after repair, as occurs in the wild type. Similar FTIR spectra also suggest that a decrease in volume by the H-to-A mutation is possibly compensated by the addition of water molecule( s). Such a modified environment is sufficient for the repair function that is probably controlled by proton-coupled electron transfer between the enzyme and substrate. On the other hand, two histidines must work in a concerted manner in the active center of the wild-type enzyme, which significantly raises the repair efficiency.

14.
Front Physiol ; 5: 179, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24847281

RESUMO

The fruit fly Drosophila melanogaster responds behaviorally to sound, gravity, and wind. Johnston's organ (JO) at the antennal base serves as a sensory organ in the fruit fly to detect these mechanosensory stimuli. Among the five anatomically defined subgroups of sensory neurons in JO, subgroups A and B detect sound vibrations and subgroups C and E respond to static deflections, such as gravity and wind. The functions of subgroup-D JO neurons, however, remain unknown. In this study, we used molecular-genetic methods to explore the physiologic properties of subgroup-D JO neurons. Both vibrations and static deflection of the antennal receiver activated subgroup-D JO neurons. This finding clearly revealed that zone D in the antennal mechanosensory and motor center (AMMC), the projection target of subgroup-D JO neurons, is a primary center for antennal vibrations and deflection in the fly brain. We anatomically identified two types of interneurons downstream of subgroup-D JO neurons, AMMC local neurons (AMMC LNs), and AMMC D1 neurons. AMMC LNs are local neurons whose projections are confined within the AMMC, connecting zones B and D. On the other hand, AMMC D1 neurons have both local dendritic arborizations within the AMMC and descending projections to the thoracic ganglia, suggesting that AMMC D1 neurons are likely to relay information of the antennal movement detected by subgroup-D JO neurons from the AMMC directly to the thorax. Together, these findings provide a neural basis for how JO and its brain targets encode information of complex movements of the fruit fly antenna.

15.
Methods Mol Biol ; 1146: 361-76, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24764098

RESUMO

Light-induced difference Fourier transform infrared (FTIR) spectroscopy is a powerful, sensitive, and informative method to study structure-function relationships in photoreceptive proteins. Strong absorption of water in the IR region is always problematic in this method, but if water content in the sample is controlled during measurements, this method can provide useful information on a single protein-bound water molecule. We established three kinds of sample preparations: hydrated film, redissolved sample, and concentrated solution. Hydrated films were used for the measurements of LOV and BLUF domains, where accurate difference FTIR spectra were obtained in the whole mid-IR region (4,000-800 cm(-1)). Vibrations of S-H stretch of cysteine, O-H stretch of water, and O-H stretch of tyrosine provide important information on hydrogen bonds in these proteins. Redissolved samples were used for the measurements of (6-4) photolyase, in which enzymatic turnover takes place. From the illumination time-dependence of excess amount of substrate, it is possible to isolate the signal originating from the binding of enzyme to substrate. If proteins are less tolerant to drying, as for example cryptochromes of the DASH type, concentrated solution is used. Detailed methodological aspects in light-induced difference FTIR spectroscopy are reviewed by mainly focusing on our results.


Assuntos
Flavinas/química , Fotorreceptores Microbianos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Flavinas/metabolismo , Fotorreceptores Microbianos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Espectroscopia de Infravermelho com Transformada de Fourier/instrumentação , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
16.
PLoS One ; 8(9): e74289, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086330

RESUMO

During courtship, many animals, including insects, birds, fish, and mammals, utilize acoustic signals to transmit information about species identity. Although auditory communication is crucial across phyla, the neuronal and physiologic processes are poorly understood. Sound-evoked chaining behavior, a display of homosexual courtship behavior in Drosophila males, has long been used as an excellent model for analyzing auditory behavior responses, outcomes of acoustic perception and higher-order brain functions. Here we developed a new method, termed ChaIN (Chain Index Numerator), in which we use a computer-based auto detection system for chaining behavior. The ChaIN system can systematically detect the chaining behavior induced by a series of modified courtship song playbacks. Two evolutionarily related Drosophila species, Drosophila melanogaster and Drosophila simulans, exhibited dramatic selective increases in chaining behavior when exposed to specific auditory cues, suggesting that auditory discrimination processes are involved in the acceleration of chaining behavior. Prolonged monotonous pulse sounds containing courtship song components also induced high intense chaining behavior. Interestingly, the chaining behavior was gradually suppressed over time when song playback continued. This behavioral change is likely to be a plastic behavior and not a simple sensory adaptation or fatigue, because the suppression was released by applying a different pulse pattern. This behavioral plasticity is not a form of habituation because different modality stimuli did not recover the behavioral suppression. Intriguingly, this plastic behavior partially depended on the cAMP signaling pathway controlled by the rutabaga adenylyl cyclase gene that is important for learning and memory. Taken together, this study demonstrates the selectivity and behavioral kinetics of the sound-induced interacting behavior of Drosophila males, and provides a basis for the systematic analysis of genes and neural circuits underlying complex acoustic behavior.


Assuntos
Comunicação Animal , Drosophila/fisiologia , Animais , Masculino , Comportamento Sexual Animal
17.
Biochemistry ; 51(29): 5774-83, 2012 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-22747528

RESUMO

Photolyases (PHRs) are blue light-activated DNA repair enzymes that maintain genetic integrity by reverting UV-induced photoproducts into normal bases. The flavin adenine dinucleotide (FAD) chromophore of PHRs has four different redox states: oxidized (FAD(ox)), anion radical (FAD(•-)), neutral radical (FADH(•)), and fully reduced (FADH(-)). We combined difference Fourier-transform infrared (FTIR) spectroscopy with UV-visible spectroscopy to study the detailed photoactivation process of Xenopus (6-4) PHR. Two photons produce the enzymatically active, fully reduced PHR from oxidized FAD: FAD(ox) is converted to semiquinone via light-induced one-electron and one-proton transfers and then to FADH(-) by light-induced one-electron transfer. We successfully trapped FAD(•-) at 200 K, where electron transfer occurs but proton transfer does not. UV-visible spectroscopy following 450 nm illumination of FAD(ox) at 277 K defined the FADH(•)/FADH(-) mixture and allowed calculation of difference FTIR spectra among the four redox states. The absence of a characteristic C=O stretching vibration indicated that the proton donor is not a protonated carboxylic acid. Structural changes in Trp and Tyr are suggested by UV-visible and FTIR analysis of FAD(•-) at 200 K. Spectral analysis of amide I vibrations revealed structural perturbation of the protein's ß-sheet during initial electron transfer (FAD(•-) formation), a transient increase in α-helicity during proton transfer (FADH(•) formation), and reversion to the initial amide I signal following subsequent electron transfer (FADH(-) formation). Consequently, in (6-4) PHR, unlike cryptochrome-DASH, formation of enzymatically active FADH(-) did not perturb α-helicity. Protein structural changes in the photoactivation of (6-4) PHR are discussed on the basis of these FTIR observations.


Assuntos
Desoxirribodipirimidina Fotoliase/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Animais , Desoxirribodipirimidina Fotoliase/química , Flavina-Adenina Dinucleotídeo/química , Luz , Oxirredução , Estrutura Secundária de Proteína , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Proteínas de Xenopus/química
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