Reply to the 'Comment on "Metal-organic green dye: chemical and physical insight into a modified Zn-benzoporphyrin for dye-sensitized solar cells"' by R. Steer, RSC Advances, 2018, DOI: 10.1039/c8ra00213d.

The authors reply to the comment by R. P. Steer discussing the reasons for their incorrect assignment of the luminescence decay of the novel compound 5,10,15-(triphenyl),20-[ethynyl-(4-carboxy)phenyl]tetrabenzoporphyrinate Zn(ii) (PETBP). Further DFT and TDDFT calculations have been performed on the compound to investigate the possibility of a direct S2-S0 decay instead of a S2-S1 conversion with a subsequent emission to the ground state. In addition, the presence of traces of very luminescent contaminants of the ring-opened type has been considered on the grounds of calculated absorption and fluorescence spectra. The results of these investigations confirm that the S2-S0 emission reported in the commented paper is not attributable to the target molecule but rather to a neglected luminescent impurity.

Cyclical regulation of GnRH gene expression in GT1-7 GnRH-secreting neurons by melatonin.

The pineal hormone melatonin plays an important role in the neuroendocrine control of reproductive physiology, but its effects on hypothalamic GnRH neurons are not yet known. We have found that GT1-7 GnRH-secreting neurons express membrane-bound G protein-coupled melatonin receptors, mt1 (Mel-1a) and MT2 (Mel-1b) as well as the orphan nuclear receptors ROR alpha and RZR beta. Melatonin (1 nM) significantly downregulates GnRH mRNA levels in a 24-h cyclical manner, an effect that is specifically inhibited by the melatonin receptor antagonist luzindole (10 microM). Repression of GnRH gene expression by melatonin appears to occur at the transcriptional level and can be mapped to the GnRH neuron-specific enhancer located within the 5' regulatory region of the GnRH gene. Using transient transfection of GT1-7 cells, downregulation of GnRH gene expression by melatonin was further localized to five specific regions within the GnRH enhancer including -1827/-1819, -1780/-1772, -1746/-1738, -1736/-1728, and -1697/-1689. Interestingly, the region located at -1736/-1728 includes sequences that correspond to two direct repeats of hexameric consensus binding sites for members of the ROR/RZR orphan nuclear receptor family. To begin to dissect the mechanisms involved in the 24-h cyclical regulation of GnRH transcription, we have found that melatonin (10 nM) induces rapid internalization of membrane-bound mt1 receptors through a beta-arrestin 1-mediated mechanism. These results provide the first evidence that melatonin may mediate its neuroendocrine control on reproductive physiology through direct actions on the GnRH neurons of the hypothalamus, both at the level of GnRH gene expression and through the regulation of G protein-coupled melatonin receptors.

Photoresponsive polypeptides.

Polypeptides containing azobenzene or spiropyran units attached to the macromolecules respond to light or dark conditions giving reversible variations of their structure. In this Account we provide a short overview of current research in the field and describe the most significant experimental examples of photoresponse effects. They include photoinduced random coil/alpha-helix transitions, helix-sense reversal, photostimulated aggregation/disaggregation processes, and photomechanical effects. These fascinating properties suggest that photoresponsive polypeptides may become suitable materials for designing sensors and devices that can be photomodulated. Findings also demonstrate that it is possible to synthesize model systems which respond to light similarly to naturally occurring photoreceptors.

The uptake, location and fluorescence of hypericin in bovine intact lens.

PURPOSE: To determine the uptake, location and fluorescence of hypericin, the active ingredient in St. John's Wort, in situ in the isolated intact calf lens. METHODS: The absorption and fluorescence spectra of hypericin 10(-5 ) M were measured in DMSO/phosphate buffer, pH 7.4) [PBS] (1/10 in volume) in the presence of alpha-crystallin (0.5 and 1.1 mg/ml). Bovine lenses were incubated in the dark for 24 hours in 10(-4) M hypericin in a DMSO/PBS (1/10 in volume) mixture. Diffused hypericin fluorescence emission was detected with a fluorescence stereomicroscope from the PBS washed lens surface. A lens-holder specially built for front-surface excitation-detection was used to measure fluorescence emission and excitation spectra of intact lenses incubated with hypericin solutions. RESULTS: As increasing concentrations of alpha-crystallin were added, the absorption and fluorescence spectra of hypericin in DMSO/PBS (1/10 in volume) changed, indicating a binding between the chromophore and the lens protein. Fluorescence emission spectra detected from the lens surface (lambda( em) = 601 and 651 nm; lambda(exc) = 550 nm) confirmed that hypericin does bind to the ocular tissues. CONCLUSIONS: The results we obtained in simplified model systems can provide clues to investigate the effects of hypericin on lens properties in physiological conditions. Hypericin could in fact bind to lens protein thus increasing the retention time of hypericin in the eye and possibly altering a-crystallin properties as a chaperone. Should therefore hypericin be taken up by the lens, this can be detected, non-invasively by its fluorescence. Therefore, ophthalmologists may use a slit-lamp or scanning fluorometry to monitor the uptake of hypericin in the eyes of patients using St. John's Wort or receiving high doses of hypericin while undergoing photodynamic therapy.

Estrogen directly respresses gonadotropin-releasing hormone (GnRH) gene expression in estrogen receptor-alpha (ERalpha)- and ERbeta-expressing GT1-7 GnRH neurons.

Estrogen has wide-ranging and complex effects on the reproductive axis, which are often difficult to interpret from in vivo studies. Estrogen negatively regulates tonic GnRH synthesis and also plays a pivotal role in the positive regulation of GnRH necessary for the preovulatory surge. To dissect the mechanisms by which these divergent effects occur, we attempted to observe the direct action of estrogen on the regulation of GnRH messenger RNA (mRNA) levels using the well characterized, GnRH-secreting, hypothalamic cell line, GT1-7. Using RT-PCR, we first investigated estrogen receptor transcript expression in GT1-7 neurons. We found that the GT1-7 cells express both estrogen receptor-alpha (ERalpha) and the recently described ERbeta mRNAs. We also detected the presence of both receptor subtypes in the GT1-7 neurons by Western blot analysis using specific ER antibodies. By Northern blot analysis of total GT1-7 RNA, we found that 17beta-estradiol (1 nM) down-regulates GnRH mRNA levels to approximately 55% of basal levels over a 48-h time course. This effect appears to occur specifically through an ER-mediated mechanism, as ICI 182,780, a complete ER antagonist, blocks the repression of GnRH mRNA levels by estradiol. The recently reported ERalpha-specific agonist/ERbeta-specific antagonist 2,2-bis-(p-hydroxyphenyl-1,1,1-trichloroethane (HPTE), a methoxychlor metabolite, also down-regulated GnRH gene expression. The repression of GnRH mRNA levels appears to occur at the transcriptional level, as simian virus 40 T antigen mRNA expression, which is under the control of 2.3 kb of the rat GnRH 5'-regulatory region, mimics the down-regulation of GnRH after treatment with estradiol. As the rat GnRH regulatory region in GT1-7 neurons does not appear to harbor a classic estrogen response element, the mechanism involved in the repression of GnRH has yet to be determined. These results suggest that estradiol directly regulates GnRH gene expression at the level of the GnRH neuron and may exert its neuroendocrine control through direct interaction with specific receptors expressed in these cells.

Photochromic polypeptides as synthetic models of biological photoreceptors: a spectroscopic study.

L-Glutamic acid polypeptides containing photochromic nitrospiropyran bound to the side chains at various percentages ("local" concentration) have been synthesized and investigated as possible artificial models of biological photoreceptors. Absorption and fluorescence spectroscopy have been utilized to investigate the photophysical and photochemical properties of nitrospiropyrans, both inserted in the polypeptide chain and in solution as "free" dye. Conformational variations produced by dark storage and light exposure of the photochromic polypeptides have been studied by means of circular dichroism. Dark-kept "free" dyes in hexafluoro-2-propanol solution in the merocyanine form ("open" form) give rise to molecular aggregates, which have been characterized as merocyanine dimers. The equilibrium constant between the monomer and the dimer, K, and their molar extinction coefficients, epsilon, at several wavelengths have been determined. Fluorescence measurements on "free" and polypeptide-bound nitrospiropyrans suggest that the dimerization process between merocyanines is favored when the photochromic units are inserted in the polypeptide chain and that under these conditions an efficient energy transfer from the monomer (donor) to the dimer (acceptor) occurs. By varying "local" as well as total nitrospiropyran concentration, it has been shown that the dimeric species result from intermolecular interactions between photochromic groups inserted in the same polypeptide chain. The alpha-helix --> random coil transition of the polypeptide structure after dark storage has eventually been shown to be the result of the dimerization process and not of the dark isomerization per se from the "closed" spiropyran form to the "open" merocyanine form of the dye.

Liquid chromatographic determination of nitrofuran residues in bovine muscle tissues.

A liquid chromatographic (LC) method was developed and statistically validated for simultaneous determination of nitrofurazone, nitrofurantoin, furazolidone, and furaltadone residues in bovine muscle tissues. These antimicrobial residues in samples stabilized at pH 6.0 were extracted with acetonitrile and purified by liquid-liquid partition between dichloromethane-ethyl acetate and hexane saturated with acetonitrile. The acetonitrile-ethyl acetate extract was concentrated, and drug residues were dissolved in LC mobile phase, filtered, and determined by LC. A C18 reversed-phase (ODS Hypersil) column at 35 degrees C, a mobile phase of 0.01M sodium acetate buffer (pH 4.5)-acetonitrile (70 + 30), and a UV/visible diode array detector at 365 nm were used. The retention times and UV spectra of peaks in spiked samples were compared with those of known nitrofurans. Limits of detection (LD) and quantitation (LQ) were 1 and 2 micrograms/kg, respectively. Average recoveries were 76% (range, 60-110%). Relative standard deviations ranged from 6 to 18% at 5 fortification levels from 1.5 to 20 micrograms/kg). (Fortification levels for furaltadone were 3 to 40 micrograms/kg). The method was used to analyze 350 samples per year from 1993 to 1995.

Spectroscopic and photoacoustic studies of hypericin embedded in liposomes as a photoreceptor model.

In photoresponsive ciliates, like Blepharisma japonicum and Stentor coeruleus, the photoreceptor pigments responsible for photomotile reactions are hypericin-type chromophores packed in highly osmiophilic subpellicular granules. Lipopsomes loaded with hypericin can constitute a simple model system, appropriate for understanding the primary light-induced molecular events triggering the sensory chain in these microorganisms. Optical absorption, steady-state and time-resolved fluorescence and pulsed photoacoustic calorimetry have been used to measure spectral distributions, fluorescence lifetimes, radiative and radiationless transition quantum yields of hypericin when assembled into egg L-alpha-phosphatidylcholine liposomes. With respect to hypericin ethanol solutions, both absorption and fluorescence maxima are 5 nm red shifted when the pigment is inserted into the lipidic microenvironment, regardless of the hypericin local concentration. Increasing by 100 times the hypericin local concentration decreases the relative fluorescence quantum yield by a factor of around 150 and the fraction of thermally released energy, conversely, increases from 0.6 to 0.9. From the analysis of fluorescence lifetimes and their relative amplitudes it appears that a subnanosecond living component is predominant at the highest hypericin local concentrations.

[Disease of the fins of Rhamdia sapo. Isolation of the etiological agents and experimental infection]. / Enfermedad de las aletas de Rhamdia sapo. Aislamiento de los agentes etiológicos e infección experimental.

During intensive culture of South-American catfish, Rhamdia sapo fingerlings an epizootic similar to fin rot was developed. Aeromonas hydrophila and Pseudomonas aeruginosa were isolated and they were treated and controlled with daily short dip, 1 ppm Furanace. The disease was experimentally reproduced and we concluded that it is result of interaction of unfavorable environment conditions such as: low dissolved oxygen, increase of solids in suspension; host condition and the presence of Aeromonas hydrophila and Pseudomonas aeruginosa.

Enfermedad de las aletas de Rhamdia sapo. Aislamiento de los agentes etiológicos e infección experimental. / [Disease of the fins of Rhamdia sapo. Isolation of the etiological agents and experimental infection]

During intensive culture of South-American catfish, Rhamdia sapo fingerlings an epizootic similar to fin rot was developed. Aeromonas hydrophila and Pseudomonas aeruginosa were isolated and they were treated and controlled with daily short dip, 1 ppm Furanace. The disease was experimentally reproduced and we concluded that it is result of interaction of unfavorable environment conditions such as: low dissolved oxygen, increase of solids in suspension; host condition and the presence of Aeromonas hydrophila and Pseudomonas aeruginosa.

Hidrolisis "in vitro" de un ester de ampicilina. I. Valoracion microbiologica. / Hydrolysis in vitro of an ampicillin ester. I. Microbiological assay

Se valora microbiologicamente con una cepa de Sarcina lutea ATCC 9341 la actividad del producto de hidrolisis de un ester de ampicilina (phthalidyl-D- alfa -aminobenzyl - penicillinate hydrochloride) (E.A.).En las experiencias "in vitro" se utilizan los siguientes parametros: pH, tiempos, temperaturas, concentraciones, procesos quimicos y enzimaticos (suero, plasma y sangre total de distintas especies animales) con y sin agitacion. Los mejores datos se obtienen sometiendo la droga a pH 8.0 con 5% de sangre humana, a 37 graus C.+/- 1 grau C, durante 90 minutos, con agitacion

Hidrolisis "in vitro" de un ester de ampicilina. II. Pruebas de sensibilidad de microorganismos de origen humano. / Hydrolysis \"in vitro\" of an ampicillin ester. II. Sensitivity test of microorganisms of human origin

Un hidrolizado de ester de ampicilina se utiliza para investigar la sensibilidad de bacterias Gran negativas y de Staphylococcus aureus, en medio liquido en rango de concentracion de 6 a 14 microgramo/ml.Tambien se realizan pruebas de difusion en agar con discos impregnados con 10 microgramos. El producto de hidrolisis conserva su actividad durante dos semanas a 4 grados C. +/- 1 grado C. Se observa correlacion de datos con ampicilina sodica estander en medio liquido y solido. Los porcentajes de sensibilidad son similares a los obtenidos por otros autores

Estudio comparativo de actividades y errores en valoraciones microbiologicas de antibioticos. / Comparative study of activities and errors in microbiological assays of antibiotics

Se valoran microbiologicamente dos aminoglucidos: sulfato de neomicina (S.N.) y sulfato de gentamicina (S.G.) por tres metodos denominados - dos por dos (2 x 2), tres por tres (3 x 3) y cinco por uno (5 x 1). Se analizan las potencias resultantes y los porcentajes de errores. En S.N.no se obtienen potencias similares con los tres metodos, en cuanto al error los valores en porcentaje son mayores para el (2 x 2) mientras que con los metodos (3 x 3) y (5 x 1) los datos son semejantes. Con S.G.no hay diferencias significativas en lo referente a potencias sin embargo los porcentajes de errores son mayores para el (2 x 2) el (5 x 1) proporciona valores intermedios y el (3 x 3) brinda los mejores datos analiticos