Photoreceptive systems in ascidians

The brain vesicle of the tadpole larva of ascidians, simple basal chordates, contains an eye-spot (ocellus), which is responsible for the photic swimming behavior. Ascidian adults also exhibit several types of light-responsive behaviors. Molecular phylogenetic studies have suggested that ascidians are the closest living relatives of vertebrates, and therefore, understanding the photoreceptive systems in ascidians is a key to uncover the origin and evolution of the vertebrate eyes. The ocellus of the ascidian larva has ciliary photoreceptors resembling those of the retina and pineal eye of vertebrates. Recent studies have indicated that the ascidian larva has phototransduction and visual cycle systems similar to those of vertebrate eyes. Comparative studies on photoreceptor systems between ascidians and vertebrates provide us clues to reconstructing the evolutionary pathway leading to the lateral and median eyes of vertebrates.     

Advances in zebrafish high content and high throughput technologies

The zebrafish has emerged as an excellent transitional screening model system between cell-based assays, which are rapid and inexpensive but have limited physiological relevance, and higher vertebrate models, which have better physiological relevance, but are more time-consuming and expensive to deploy. As vertebrates, zebrafish maintain significant evolutionary proximity to humans and have been validated as robust models for drug research, studies of mechanism and behavioral genetics. Unlike higher vertebrate models, zebrafish are well-suited to high-throughput applications owing to their high fecundity, rapid extrauterine development and transparency during organogenesis enabling in vivo labeling and imaging. Recent advances have been made in automating high content and high-throughput zebrafish screens, with the goal of developing fully automated drug screening platforms. The application and continued development of these technologies holds potential clinical significance in drug discovery and elucidating disease mechanisms.     

Analysis of melatonin, 5-methoxytryptophol and 5-methoxyindoleacetic acid in the pineal gland and retina of hamster by capillary column gas chromatography-mass spectrometry

A specific capillary column gas chromatographic-mass spectrometric method was used to determine 5-methoxyindoles in the pineal gland and retina of the golden hamster during a light-dark (14:10) cycle. In the pineal gland, the mean levels of melatonin ranged from 0.15 to 2.4 pmol per gland, with a maximum in the dark. The levels of 5-methoxytryptophol and 5-methoxyindoleacetic acid were in the same range, but peaked during light. In the retina the levels of melatonin were about 100 pmol/g, and seemed not to differ between light and dark. The level of 5-methoxyindoleacetic acid were in the same range during light but were below the detection limit during dark.     

Enantioselective micro-2D-HPLC determination of aspartic acid in the pineal glands of rodents with various melatonin contents

Enantioselective determination of aspartic acid (Asp) in the pineal gland of rodents with various melatonin contents was performed using a highly sensitive and selective two-dimensional HPLC system. After derivatization of the amino group with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), NBD-Asp was separated using a capillary monolithic ODS column in the first dimension. The fraction of NBD-Asp was automatically collected and transferred to the second dimension, and the D- and L-Asp were separated and determined using a narrowbore enantioselective column. Large amounts of D-Asp were observed in the pineal gland of the rats and specific strains of mice (C3H and CBA) possessing a high concentration of melatonin in their pineal gland. On the other hand, the amounts of D-Asp were small in the pineal gland of mice possessing a trace or no melatonin in their pineal gland (ddY, ICR, C57BL and BALB/c). In other tissues and physiological fluids, no significant strain-dependent changes of the D-Asp amounts were observed. These results indicate that large amounts of D-Asp are present only in the pineal gland containing large amounts of melatonin, and special care should be taken when selecting mouse strains for the investigation of D-Asp.     

Characterization of Lamprey Rhodopsin by Isolation from Lamprey Retina and Expression in Mammalian Cells

Abstract— A visual pigment was extracted from lamprey retina and was expressed in cultured mammalian cells (293S) using a cDNA fragment isolated from lamprey retina. The extracted pigment, a putative lamprey rhodopsin, had an absorption maximum at 503 nm. The recombinant lamprey rhodopsin, reconstituted with 11- cis -retinal, showed an absorption maximum at about 500 nm. Both pigments reacted with an anti-bovine rhodopsin antibody (Rh29), which recognizes the short photoreceptor cells in lamprey retina. Unlike rhodopsins of higher vertebrates, the lamprey rhodopsin bleached gradually in the presence of 100 m M hydroxylamine even in the dark. Our results suggest that, despite its high similarities with other vertebrate rhodopsins, lamprey rhodopsin has a character different from those of higher vertebrates.     

Controlling the biological effects of spermine using a synthetic receptor

Polyamines play an important role in biology, yet their exact function in many processes is poorly understood. Artificial host molecules capable of sequestering polyamines could be useful tools for studying their cellular function. However, designing synthetic receptors with affinities sufficient to compete with biological polyamine receptors remains a huge challenge. Binding affinities of synthetic hosts are typically separated by a gap of several orders of magnitude from those of biomolecules. We now report that a dynamic combinatorial selection approach can deliver a synthetic receptor that bridges this gap. The selected receptor binds spermine with a dissociation constant of 22 nM, sufficient to remove it from its natural host DNA and reverse some of the biological effects of spermine on the nucleic acid. In low concentrations, spermine induces the formation of left-handed DNA, but upon addition of our receptor, the DNA reverts back to its right-handed form. NMR studies and computer simulations suggest that the spermine complex has the form of a pseudo-rotaxane. The spermine receptor is a promising lead for the development of therapeutics or molecular probes for elucidating spermine's role in cell biology.     

老年痴呆症的分子病理机制

老年痴呆症是中枢神经退化性疾病 ,被认为是危害人类健康的一大综合症。因为老年痴呆症不仅具有多重分子病理而且是慢性综合症 ,所以对于它的病因及生化机理很难达成一致。本文主要阐述了一些被普遍接受的分子病变机理和研究现状 ,并简单介绍部分有效或潜在的治疗方法     

Gq-coupled rhodopsin subfamily composed of invertebrate visual pigment and melanopsin

Rhodopsins (rhodopsins and their related photopigments) are phylogenetically classified into at least seven subfamilies, which are also roughly discriminated by molecular function. The Gq-coupled rhodopsin subfamily, members of which activate the Gq type G protein upon light absorption, contains pigments which underlie both visual and nonvisual physiologic functions. Gq-coupled visual pigments have been found in the rhabdomeric photoreceptor cells of varied protostomes, and those of molluskans and arthropods have been extensively investigated. Recently, a novel photopigment, melanopsin, and its homologs have been identified in varied vertebrates. In mammals, melanopsin is localized in retinal ganglion cells and is involved in nonvisual systems, including circadian entrainment and pupillary light responses. More recently, we discovered a melanopsin homolog in amphioxus, the closest living invertebrate to vertebrates. Amphioxus melanopsin is localized in putative nonvisual photoreceptor cells with rhabdomeric morphology and exhibits molecular properties almost identical to those of invertebrate Gq-coupled visual pigments. The localization and properties of amphioxus melanopsin bridged the functional and evolutionary gap between invertebrate Gq-coupled visual pigments and vertebrate circadian photopigment melanopsins. Research into the Gq-coupled rhodopsin subfamily, especially invertebrate melanopsins, will provide an opportunity to investigate the evolution of various physiologic functions, based on orthologous genes, during animal evolution.     

Effects of diazepam administration on melatonin synthesis in the rat pineal gland in vivo.

The effect of diazepam (DZP) on melatonin synthesis in rat pineal gland was investigated in vivo. Subcutaneous injection of DZP (3 mg/kg) 1 h before the start of darkness significantly suppressed nocturnal elevations of pineal N-acetylserotonin (NAS) and melatonin contents in rats, and caused a 2-h delay in reaching the maximum melatonin level in the dark phase. DZP treatment also markedly suppressed the dark-induced increase of pineal N-acetyltransferase activity, which catalyzes the rate-limiting step in melatonin synthesis, but had no effect on hydroxyindole-O-methyltransferase activity, which catalyzes the final step of melatonin formation. Pineal norepinephrine and dopamine contents, in contrast, were not altered by DZP injection. The distribution rate of DZP to the brain reached the highest level 30 min after a single injection, while that to the pineal gland was observed 5 h later (i.e., 4 h after the start of darkness). It is clear that the inhibitory effect of DZP on melatonin synthesis in rat pineal gland appears concomitantly with the increase in the distribution volume of DZP into this gland. These results suggest that the inhibitory effect of DZP on melatonin synthesis results from the drug's direct action on the rat pineal gland.     

Effects of adrenergic blockers or bicuculline on diazepam induced changes in rat pineal melatonin synthesis in vivo and in vitro.

The effect of propranolol (PPL), phenoxybenzamine (PBZ) or bicuculline (BCL) on the diazepam (DZP)-induced changes of pineal melatonin synthesis in male rats was examined in vivo and in vitro. Administration of PBZ did not affect the inhibitory action of DZP on pineal melatonin synthesis in vivo. A single injection of PPL inhibited the pineal melatonin synthesis similarly to the administration of DZP alone, but the two drugs together did not exhibit additive or synergistic effects on the melatonin synthesis. Significant decreases in the N-acetyltransferase (NAT) activity and the N-acetylserotonin (NAS) and melatonin contents were observed in the BCL-injected group, being greater than those in the DZP-treated group. Unexpectedly, however, the combination treatment of DZP and BCL causes an increase in the NAT activity and melatonin content compared with the BCL-alone group. Incubation with DZP at higher concentrations resulted in an increase of pineal NAT activity in vitro, but this increase was inhibited by preincubation with PPL, PBZ or BCL. DZP treatment thus appeared to have different effects on pineal NAT activity in vivo and in vitro. These results suggest that both a GABAergic mechanism and peripheral benzodiazepine (BZP) receptors in rat pineal gland may be involved in the modulation of melatonin synthesis by DZP.     

Melatonin prevents magnetic-field-induced neurite outgrowth in a subline of pheochromocytoma cells,PC12D

Exposure to extremely low frequency (ELF) magnetic fields (MFs) has been reported to affect several cellular processes, including cell growth and differentiation. Other research has demonstrated that the pineal gland and its hormone melatonin have a wide spectrum of effects in cells and organs and can exert modulatory actions on cell proliferation and cell differentiation. Since ELF electric and magnetic fields have been shown to influence pineal activity and melatonin synthesis and/or function, it has been suggested that some of the reported effects of ELF MFs could be a consequence of the direct action of these fields on the pineal gland and/or melatonin function. Possible interactions between MFs and melatonin effects are in an early stage of investigation. In this study, we have investigated the influence of melatonin on the in vitro response of a subline of pheochromocytoma cells, PC12D, to a MF. Cells were exposed to the combined action of a physiological concentration (10⁻¹⁰ M) of melatonin and a vertical, 50 Hz, 40 mG rms MF for 23 h. At the end of the treatment, the percentages of neurite-bearing cells were determined by microscopic examination and compared with those from samples treated with the field alone or with melatonin alone. MF exposure alone significantly increased the neurite outgrowth when compared with negative controls, supporting previous results by Blackman and coworkers; this effect was not observed when melatonin was present in the medium from the onset of the exposure. Although the mechanisms of action of melatonin and ELF MFs at the cellular level remain unknown, the present data suggest that physiological levels of melatonin can prevent PC12D cells from responding to the MF stimulus.     

Photochemistry and photobiology of cryptochrome blue-light photopigments: the search for a photocycle

Cryptochromes are flavoproteins that exhibit high sequence and structural similarity to the light-dependent DNA-repair enzyme, photolyase. Cryptochromes have lost the ability to repair DNA; instead, they use the energy from near-UV/blue light to regulate a variety of growth and adaptive processes in organisms ranging from bacteria to humans. The photocycle of cryptochrome is not yet known, although it is hypothesized that it may share some similarity to that of photolyase, which utilizes light-driven electron transfer from the catalytic flavin chromophore. In this review, we present genetic evidence for the photoreceptive role of cryptochromes and discuss recent biochemical studies that have furthered our understanding of the cryptochrome photocycle. In particular, the role of the unique C-terminal domain in cryptochrome phototransduction is discussed.     

Identification and quantification of phosphatidylcholines containing very-long-chain polyunsaturated fatty acid in bovine and human retina using liquid chromatography/tandem mass spectrometry

The retina is one of the vertebrate tissues with the highest content in polyunsaturated fatty acids (PUFA). A large proportion of retinal phospholipids, especially those found in photoreceptor membranes, are dipolyunsaturated molecular species. Among them, dipolyunsaturated phosphatidylcholine (PC) molecular species are known to contain very-long-chain polyunsaturated fatty acids (VLC-PUFA) from the n-3 and n-6 series having 24-36 carbon atoms (C24-C36) and four to six double bonds. Recent interest in the role played by VLC-PUFA arose from the findings that a protein called elongation of very-long-chain fatty acids 4 (ELOVL4) is involved in their biosynthesis and that mutations in the ELOVL4 gene are associated with Stargardt-like macular dystrophy (STD3), a dominantly inherited juvenile macular degeneration leading to vision loss. The aim of the present study was to develop an HPLC-ESI-MS/MS method for the structural characterisation and the quantification of dipolyunsaturated PC molecular species containing VLC-PUFA and validate this methodology on retinas from bovines and human donors. Successful separation of phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), PC, lyso-phosphatidylcholine (LPC) and sphingomyelin (SM) was achieved using a silica gel column and a gradient of hexane/isopropanol/water containing ammonium formate as a mobile phase. A complete structural characterisation of intact phosphatidylcholine species was obtained by collision-induced dissociation (CID) in the negative mode. Fatty acid composition and distribution can be clearly assigned based on the intensity of sn-2/sn-1 fragment ions. The PC species were characterised on bovine retina, 28 of which were dipolyunsaturated PC species containing one VLC-PUFA (C24-C36) with three to six double bonds. VLC-PUFA was always in the sn-1 position while PUFA at the sn-2 position was exclusively docosahexaenoic acid (DHA, C22:6n-3). Most of these VLC-PUFA-containing dipolyunsaturated PCs were detected and quantified in human retinas. The quantitative analysis of the different PC molecular species was performed in the positive mode using precursor ion scanning of m/z 184 and 14:0/14:0-PC and 24:0/24:0-PC as internal standards. The relationship between the MS peak intensities of different PC species and their carbon chain length was included for calibration. The main compounds represented were those having VLC-PUFA with 32 carbon atoms (C32:3, C32:4, C32:5 and C32:6) and 34 carbon atoms (C34:3, C34:4, C34:5 and C34:6). Dipolyunsaturated PCs with 36:5 and 36:6 were detected but in smaller quantities. In conclusion, this new HPLC-ESI-MS/MS method is sensitive and specific enough to structurally characterise and quantify all molecular PC species, including those esterified with VLC-PUFA. This technique is valuable for a precise characterisation of PC molecular species containing VLC-PUFA in retina and may be useful for a better understanding of the pathogenesis of STD3.     

Chemical tools for functional studies of glycans

In recent years a variety of chemical approaches have been developed for elucidating the molecular basis of biological processes in which glycans participate. The chemical technologies uncovered have greatly influenced the progress of glycomics research programs. This tutorial review highlights recent advances in chemical tools which have been developed and their applications in studies aimed at gaining a better understanding of the roles that glycans play in biological processes.     

Variation of melatonin and serotonin content in rat pineal gland with sex and oestrous phase difference determined by high-performance liquid chromatography with fluorimetric detection

Simultaneous determination of melatonin and serotonin in rat pineal gland is described using reversed-phase high-performance liquid chromatography with fluorimetric detection. These indoles were analysed isocratically within 15 min. In this work, veratric acid (3,4-dimethoxybenzoic acid), which has fluorescence characteristics (lambda ex = 290 nm, lambda em = 350 nm) around the wavelength of native fluorescence of melatonin (lambda ex = 285 nm, lambda em = 345 nm), was used as an internal standard. This method was applied to the determination of melatonin and serotonin in male and female rat pineal gland. No significant differences between the two groups were observed in the pineal melatonin and serotonin contents. The pineal melatonin and serotonin contents were compared with the oestrous and the di-oestrous phases of female rats. They were not widely different from each other.     

Micellar electrokinetic capillary chromatography for fast separation and sensitive determination of melatonin and related indoleamines using end-column amperometric detection

MEKC was used in conjunction with end-column amperometric detection (AD) at a carbon disc electrode (0.3 mm diameter) for the selective and sensitive determination of melatonin and its five related indoleamines including its precursors and metabolites in the pineal gland. The introduction of a sample stacking technique in injection and the buffer additive SDS in the buffer solution system provided the rapid and sensitive analysis. Optimal buffer conditions (10 mmol/L phosphate containing 20 mmol/L SDS, pH 7.2), detection potential (+1.0 V vs. Ag/AgCl), and electrokinetic injection 10 s with the separation voltage of 24 kV were employed to achieve the baseline separation of six pineal hormones within 15 min. The peak currents and the analyte concentrations have a good linear relationship over the range of 6.0 x 10(-8) 6.0 x 10(-5 )mol/L. The detection limits for six pineal hormones by AD are 9.7 to 41.8 nmol/L (equal to 2.0 to 9.7 ng/mL) (S/N = 3), respectively. It is proved to provide about 30- to 250-fold improvement over UV, and be comparable with the sensitive fluorescence detection, which needs pre-column derivatization. The proposed method has been applied for analysis of melatonin and related indoleamines in rat pineal glands. A very simple sample pretreatment procedure, merely involving the homogenization step in perchloric acid, was enough to achieve recoveries in the range of 71 to 127% for all the analytes in the pineal gland.     

Theoretical study of molecular conduction: I. Effective Green's function based on perturbation theory

There have been many experimental and theoretical studies on molecular conduction, as it is a fundamental parameter in the study of molecular‐scale electronics. We have investigated the features of molecular conduction using a Green's function method, which has often been used to solve problems in quantum transport and is also effective in elucidating electron transport in molecules. We have obtained the novel effective Green's functions, including the first‐order energy corrections, by accommodating the self‐energy of the electrodes as perturbation terms. Although these approximate Green's functions only provide information on the first‐order energy corrections, they can involve the elementary properties of molecular conduction. We propose a scheme for the analysis of the relations between molecular orbitals and their roles in molecular conduction and present analytical calculations for normal and cyclic polyenes. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2006     

Simulated molecular evolution in a full combinatorial library

BACKGROUND: The Darwinian concept of 'survival of the fittest' has inspired the development of evolutionary optimization methods to find molecules with desired properties in iterative feedback cycles of synthesis and testing. These methods have recently been applied to the computer-guided heuristic selection of molecules that bind with high affinity to a given biological target. We describe the optimization behavior and performance of genetic algorithms (GAs) that select molecules from a combinatorial library of potential thrombin inhibitors in 'artificial molecular evolution' experiments, on the basis of biological screening results. RESULTS: A full combinatorial library of 15,360 members structurally biased towards the serine protease thrombin was synthesized, and all were tested for their ability to inhibit the protease activity of thrombin. Using the resulting large structure-activity landscape, we simulated the evolutionary selection of potent thrombin inhibitors from this library using GAs. Optimal parameter sets were found (encoding strategy, population size, mutation and cross-over rate) for this artificial molecular evolution. CONCLUSIONS: A GA-based evolutionary selection is a valuable combinatorial optimization strategy to discover compounds with desired properties without needing to synthesize and test all possible combinations (i.e. all molecules). GAs are especially powerful when dealing with very large combinatorial libraries for which synthesis and screening of all members is not possible and/or when only a small number of compounds compared with the library size can be synthesized or tested. The optimization gradient or 'learning' per individual increases when using smaller population sizes and decreases for higher mutation rates.     

Enhanced efficiency of direct-space structure solution from powder X-ray diffraction data in the case of conformationally flexible molecules

A strategy is reported for assessing the feasibility of molecular conformations within direct-space structure-solution calculations of organic molecular crystal structures from powder X-ray diffraction data, focusing in particular on the genetic algorithm technique for structure solution in which fitness is defined as a function of the whole-profile figure-of-merit Rwp. The strategy employs a readily computed distance-based function to assess the feasibility of the molecular conformation in each trial structure generated in the genetic algorithm calculation, and structures considered to have low-feasibility conformations are penalized within the evolutionary process. The strategy is shown to lead to significant improvements in the success rate of structure-solution calculations in the case of flexible molecules with a significant number of conformational degrees of freedom.