GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity

Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.     

Circular dichroism of 1,3-dioxane-type (2'-naphthyl)methylene acetals of glycosides

The CD spectra are reported for a series of 1,3-dioxane-type 4,6-O-(2'-naphthyl)methylene acetals of carbohydrates with and without interacting aromatic protective groups on the C-1, C-2, and C-3 hydroxy groups. In the absence of interacting chromophores, the signs of the (1)B transitions are not sensitive to the configuration of C-4, while the signs of the weak (1)L(a) bands are opposite in the galacto and gluco derivatives. The equatorial parallel conformation is found to be the preferred conformation of the 2-naphthyl group in the solid state by X-ray diffraction. The intense (1)B(a) and (1)B(b) transitions of the naphthalene chromophore allowed a safe configurational assignment by exciton coupled interaction with the aromatic protective groups in para-methoxyphenyl-beta-D-glycosides. The origin of the observed CEs were deduced and the additivity of the interactions was studied. The direction of the hydrogenolytic cleavage of 4,6-O-(2'-naphthyl)methylene acetal of carbohydrates could also be detected by the (1)B(b) transition of the 2-naphthyl chromophore.     

Capacitation of mammalian spermatozoa in vivo, with a specific focus on events in the Fallopian tubes

This essay argues strongly that for those sperm cells involved in fertilisation, the process of capacitation represents an active and specific coordination within succeeding regions of the female tract and one whose completion is synchronised with the events of ovulation. Observations on the time-course of capacitation when spermatozoa are first exposed to the uterus and then progress to the Fallopian tubes indicate a synergistic influence of these adjoining portions of the female tract on the rate of capacitation. Three concepts on the control of capacitation are introduced to emphasise the importance of integration in vivo, namely that (1) completion of capacitation is a peri-ovulatory event, (2) suppression of completion of capacitation is an essential storage strategy during a long pre-ovulatory interval, and (3) the process of capacitation comes under the influence of local and systemic ovarian control mechanisms, especially the secretion of progesterone from Graafian follicles soon to ovulate. The last would act to coordinate the final maturation and meeting of male and female gametes. Despite the preceding points, the requirement for such integrated in vivo programming of sperm cell maturation can clearly be overridden in systems of culture. The most reasonable interpretation here would be that a microdrop of culture medium containing eggs, follicular cells and components of follicular fluid would to a considerable extent represent a post-ovulatory environment. Within such a preparation, there would be leaching of the sperm surface among the relatively vast and heterogeneous population of cells, and a proportion of spermatozoa could then respond to 'post-ovulatory signals', not least to molecular influences of the zona pellucida and vitelline products for completion of capacitation. Nonetheless, a physiologically meaningful interpretation of capacitation calls for a stepwise analysis of the dynamic interactions between sperm cell and female tract at successive stages between the uterus and ampullary-isthmic junction.     

Red-edge-excitation fluorescence spectroscopy of indole and tryptophan

Studies on the dependence of indole and tryptophan fluorescence emission spectra on excitation wavelength, ^ex, show that the emission shifts to longer wavelengths for red-edge excitation in different solid and viscous solvents. In solid systems the spectral shifts for excitation in the range from 290 to 310 nm can reach tens of nm, and they are more significant than changes of ^ex. In a viscous medium the magnitude of this effect is shown to be directly related to the dipole-reorientational relaxation of solvent molecules in the environment of the chromophore, which allows the relaxation times to be estimated. The method involves simple steady-state measurements of fluorescence spectra at the maximum and at the red edge of the absorption band. Since it is not necessary to obtain information on the fluorescence spectra of completely relaxed states, this method for the estimation of relaxation times may have advantages in studies of proteins compared with the conventional relaxation shift method, and may produce complementary information to that obtained by nanosecond time-resolved spectroscopy.     

Chromatic adaptation in a mutant of Fremyella diplosiphon incapable of phycoerythrin synthesis

A mutant of the chromatically adapting cyanobacterium Fremyella diplosiphon, incapable of phycoerythrin synthesis but responding to wavelength modulation of its biliprotein content, was isolated. The biliprotein composition of the mutant and of the wild type were identical after growth in red light, but green light induced, in the mutant, the synthesis of a biliviolin-type chromophore bound to some of the alpha subunits of its phycocyanin. Implications of the results on the regulation and possible pathways of biliprotein biosynthesis are discussed.     

Iridium oxide pH microelectrode

The manufacture, calibration, and signal conditioning during construction of an iridium/iridium oxide pH microsensor is described. The microsensor was designed to be used extracellularly, primarily in biofilm research. The sensing tip diameters were typically in the range of 3-15 mum. The iridium oxide was formed by potential cycling in dilute sulfuric acid. A pH profule across a denitrifying biofilm was measured as an example of an application. The higher Nernstian slope (70-80 mV/pH for fresh electrodes), increased rigidity, and restriction of the sensing tip to the outermost end of the electrode are features which make the iridium/iridium oxide pH microelectrode superior to a glass microelectrode. (c) 1992 John Wiley & Sons, Inc.     

Crystal Structure of Green Fluorescent Protein Clover and Design of Clover-Based Redox Sensors

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Haem oxygenase: A functionally diverse enzyme of photosynthetic organisms and its role in phytochrome chromophore biosynthesis,cellular signalling and defence mechanisms

Haem oxygenase (HO) is a universal enzyme that catalyses stereospecific cleavage of haem to BV IX α and liberates Fe⁺² ion and CO as by‐product. Beside haem degradation, it has important functions in plants that include cellular defence, stomatal regulation, iron mobilization, phytochrome chromophore synthesis, and lateral root formation. Phytochromes are an extended family of photoreceptors with a molecular mass of 250 kDa and occur as a dimer made up of 2 equivalent subunits of 125 kDa each. Each subunit is made of two components: the chromophore, a light‐capturing pigment molecule and the apoprotein. Biosynthesis of phytochrome (phy) chromophore includes the oxidative splitting of haem to biliverdin IX by an enzyme HO, which is the decisive step in the biosynthesis. In photosynthetic organisms, BVα is reduced to 3Z PΦB by a ferredoxin‐dependent PΦB synthase that finally isomerised to PΦB. The synthesized PΦB assembles with the phytochrome apoprotein in the cytoplasm to generate holophytochrome. Thus, necessary for photomorphogenesis in plants, which has confirmed from the genetic studies, conducted on Arabidopsis thaliana and pea. Besides the phytochrome chromophore synthesis, the review also emphasises on the current advances conducted in plant HO implying its developmental and defensive role.     

MICROENVIRONMENTS AND MICROSCALE PRODUCTIVITY OF CYANOBACTERIAL DESERT CRUSTS1

We used microsensors to characterize physicochemical microenvironments and photosynthesis occurring immediately after water saturation in two desert soil crusts from southeastern Utah, which were formed by the cyanobacteria Microcoleus vaginatus Gomont, Nostoc spp., and Scytonema sp. The light fields within the crusts presented steep vertical gradients in magnitude and spectral composition. Near-surface light-trapping zones were formed due to the scattering nature of the sand particles, but strong light attenuation resulted in euphotic zones only ca. 1 mm deep, which were progressively enriched in longer wavelengths with depth. Rates of gross photosynthesis (3.4–9.4 mmol O₂·m^?2·h^?1) and dark respiration (0.81–3.1 mmol O^?2·m^?2·h^?1) occurring within 1 to several mm from the surface were high enough to drive the formation of marked oxygen microenvironments that ranged from oxygen supersaturation to anoxia. The photosynthetic activity also resulted in localized pH values in excess of 10, 2–3 units above the soil pH. Differences in metabolic parameters and community structure between two types of crusts were consistent with a successional pattern, which could be partially explained on the basis of the microenvironments. We discuss the significance of high metabolic rates and the formation of microenvironments for the ecology of desert crusts, as well as the advantages and limitations of microsensor-based methods for crust investigation.