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PubMedDate not supplied

Photoreceptors and visual pigments in the red-eared turtle, Trachemys scripta elegans.

Absorbance spectra of cone outer segments and oil droplets were recorded microspectrophotometrically in the retina of the red-eared turtle, Trachemys scripta elegans. There are four cone visual pigments, with lambda(max) = 617 nm (red sensitive), 515 nm (green sensitive), 458 nm (blue sensitive), and 372 nm (UV-sensitive). The red-sensitive pigment resides in single cones with red or orange oil droplets, and in both members of double cones. The principal member of the double cone contains an orange oil droplet, and the accessory member is droplet free. The green-sensitive pigment is situated in single cones with orange/dark yellow droplets. The blue-sensitive pigment is combined with the UV-absorbing oil droplet in single cones. The UV-sensitive pigment resides in single cones with clear oil droplets that exhibited virtually no absorbance down to 325 nm. Thus, seven types of cones can be identified based on their morphology, oil droplet color, and the visual pigment absorbance. At the moment, this is the most complex cone system described for vertebrates.

Animals↗
PubMedDate not supplied

Visual pigments and dichroism of anchovy cones: a model system for polarization detection.

The retinas of anchovies have two unique photoreceptor types: "bifid" and "long" cones (Fineran & Nicol, 1976). The outer segments of these cells contain multiple layers of membranes (lamellae) oriented longitudinally (axially). This orientation is distinct from that in all other vertebrate rods and cones, where the lamellae are stacked transversely with their planes perpendicular to the incident light path. Although the common arrangement provides optimal absorption for normally incident light rays, it is also insensitive to the rays' direction of vibration (i.e. their polarization). In contrast, the two mutually perpendicular sets of axially oriented lamellae segregated into bifid and long cones could function as the principal analyzers for linearly polarized light, as previously hypothesized (Fineran & Nicol, 1976, 1978). Here, we report on a microspectrophotometric study that shows (1) the presence of two spectrally distinct visual pigments in the three photoreceptor types of the bay anchovy retina; these are typical vertebrate pigments in that they bleach, when exposed to light, and have absorption spectra like all other vitamin A1-based visual pigments; (2) that the rods and cones exhibit dichroic absorption of light in accordance with their lamellar orientation, and (3) that the two cone types of the retina contain a spectrally indistinguishable pigment with peak absorbance (lambda(max)) around 540 nm, while the rods contain a rhodopsin-like pigment with lambda(max) near 500 nm. Compared to other vertebrates, anchovies are remarkable for using a monochromatic cone system with unusual specializations supportive of a polarization detection system.

Animals↗
PubMed2000

Characterization of the primary photointermediates of Drosophila rhodopsin.

Invertebrate opsins are unique among the visual pigments because the light-activated conformation, metarhodopsin, is stable following exposure to light in vivo. Recovery of the light-activated pigment to the dark conformation (or resting state) occurs either thermally or photochemically. There is no evidence to suggest that the chromophore becomes detached from the protein during any stage in the formation or recovery processes. Biochemical and structural studies of invertebrate opsins have been limited by the inability to express and purify rhodopsins for structure-function studies. In this study, we used Drosophila to produce an epitope-tagged opsin, Rh1-1D4, in quantities suitable for spectroscopic and photochemical characterization. When expressed in Drosophila, Rh1-1D4 is localized to the rhabdomere membranes, has the same spectral properties in vivo as wild-type Rh1, and activates the phototransduction cascade in a normal manner. Purified Rh1-1D4 visual pigment has an absorption maximum of the dark-adapted state of 474 nm, while the metarhodopsin absorption maximum is 572 nm. However, the metarhodopsin state is not stable as purified in dodecyl maltoside but decays with kinetics that require a double-exponential fit having lifetimes of 280 and 2700 s. We investigated the primary properties of the pigment at low temperature. At 70 K, the pigment undergoes a temperature-induced red shift to 486 nm. Upon illumination with 435 nm light, a photostationary state mixture is formed consisting of bathorhodopsin (lambda(max) = 545 nm) and isorhodopsin (lambda(max) = 462 nm). We also compared the spectroscopic and photochemical properties of this pigment with other vertebrate pigments. We conclude that the binding site of Drosophila rhodopsin is similar to that of bovine rhodopsin and is characterized by a protonated Schiff base chromophore stabilized via a single negatively charged counterion.

Animals↗
PubMed2000

Structural basis for substrate specificity differences of horse liver alcohol dehydrogenase isozymes.

A structure determination in combination with a kinetic study of the steroid converting isozyme of horse liver alcohol dehydrogenase, SS-ADH, is presented. Kinetic parameters for the substrates, 5beta-androstane-3beta,17beta-ol, 5beta-androstane-17beta-ol-3-one, ethanol, and various secondary alcohols and the corresponding ketones are compared for the SS- and EE-isozymes which differ by nine amino acid substitutions and one deletion. Differences in substrate specificity and stereoselectivity are explained on the basis of individual kinetic rate constants for the underlying ordered bi-bi mechanism. SS-ADH was crystallized in complex with 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan -24-acid (cholic acid) and NAD(+), but microspectrophotometric analysis of single crystals proved it to be a mixed complex containing 60-70% NAD(+) and 30-40% NADH. The crystals belong to the space group P2(1) with cell dimensions a = 55.0 A, b = 73.2 A, c = 92.5 A, and beta = 102.5 degrees. A 98% complete data set to 1.54-A resolution was collected at 100 K using synchrotron radiation. The structure was solved by the molecular replacement method utilizing EE-ADH as the search model. The major structural difference between the isozymes is a widening of the substrate channel. The largest shifts in C(alpha) carbon positions (about 5 A) are observed in the loop region, in which a deletion of Asp115 is found in the SS isozyme. SS-ADH easily accommodates cholic acid, whereas steroid substrates of similar bulkiness would not fit into the EE-ADH substrate site. In the ternary complex with NAD(+)/NADH, we find that the carboxyl group of cholic acid ligates to the active site zinc ion, which probably contributes to the strong binding in the ternary NAD(+) complex.

Alcohol Dehydrogenase↗
PubMed2003

Protein and DNA residue orientations in the filamentous virus Pf1 determined by polarized Raman and polarized FTIR spectroscopy.

The Pseudomonas bacteriophage Pf1 is a long ( approximately 2000 nm) and thin ( approximately 6.5 nm) filament consisting of a covalently closed, single-stranded DNA genome of 7349 nucleotides coated by 7350 copies of a 46-residue alpha-helical subunit. The coat subunits are arranged as a superhelix of C(1)()S(5.4)() symmetry (class II). Polarized Raman and polarized FTIR spectroscopy of oriented Pf1 fibers show that the packaged single-stranded DNA genome is ordered specifically with respect to the capsid superhelix. Bases are nonrandomly arranged along the capsid interior, deoxynucleosides are uniformly in the C2'-endo/anti conformation, and the average DNA phosphodioxy group (PO(2)(-)) is oriented so that the line connecting the oxygen atoms (O.O) forms an angle of 71 degrees +/- 5 degrees with the virion axis. Raman and infrared amide band polarizations show that the subunit alpha-helix axis is inclined at an average angle of 16 degrees +/- 4 degrees with respect to the virion axis. The alpha-helical symmetry of the capsid subunit is remarkably rigorous, resulting in splitting of Raman-active helix vibrational modes at 351, 445 and 1026 cm(-)(1) into apparent A-type and E(2)()-type symmetry pairs. The subunit tyrosines (Tyr 25 and Tyr 40) are oriented with phenoxyl rings packed relatively close to parallel to the virion axis. The Tyr 25 and Tyr 40 orientations of Pf1 are surprisingly close to those observed for Tyr 21 and Tyr 24 of the Ff virion (C(5)()S(2)() symmetry, class I), suggesting a preferred tyrosyl side chain conformation in packed alpha-helical subunits, irrespective of capsid symmetry. The polarized Raman spectra also provide information on the orientations of subunit alanine, valine, leucine and isoleucine side chains of the Pf1 virion.

Alanine↗
PubMed2003

Polarized absorption spectra of green fluorescent protein single crystals: transition dipole moment directions.

Polarized absorption spectra of orthorhombic crystals of wild-type green fluorescent protein (GFP) were measured between 350 and 520 nm to obtain information on the directions of the electronic transition dipole moments ((-->)m) of the chromophore relative to the molecular axes. The transition dipole moment orientation is a basic spectroscopic parameter of relevance to biologists when interpreting Förster-type fluorescence resonance energy transfer data and for comparing absorbance and fluorescence spectra of GFP with quantum chemical calculations. Maximal extinction was obtained throughout the spectrum when the polarization direction of the electric vector of incident light was parallel to the c-axis of the crystal. The transition dipole moments were assumed to be parallel to the plane of the chromophore. With this assumption and the measured dichroic ratios in the crystals, the transition dipole moments associated with the neutral (lambda(max) = 398 nm) and anionic (lambda(max) = 478 nm) forms of the chromophore were found to subtend angles of approximately 26 degrees and 13 degrees (counterclockwise), respectively, with the vector that joins the phenolic and imidazolinone oxygen atoms of the chromophore.

Amino Acid Substitution↗
PubMed1998

Conformational changes occurring upon reduction and NO binding in nitrite reductase from Pseudomonas aeruginosa.

Nitrite reductase (NiR) from Pseudomonas aeruginosa (EC 1.9.3.2) (NiR-Pa) is a soluble enzyme catalyzing the reduction of nitrite (NO2-) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which each monomer carries one c and one d1 heme. The oxidized and reduced forms of NiR from Paracoccus denitrificans GB17 (previously called Thiosphaera pantotropha) (NiR-Pd) have been described [Fülop, V., et al. (1995) Cell 81, 369-377; Williams, P. A., et al. (1997) Nature 389, 406-412], and we recently reported on the structure of oxidized NiR-Pa at 2.15 A [Nurizzo, D., et al. (1997) Structure 5, 1157-1171]. Although the domains carrying the d1 heme are almost identical in both NiR-Pa and NiR-Pd oxidized and reduced structures, the c heme domains show a different pattern of c heme coordination, depending on the species and the redox state. The sixth d1 heme ligand in oxidized NiR-Pd was found to be Tyr25, whereas in NiR-Pa, the homologuous Tyr10 does not interact directly with Fe3+, but via a hydroxide ion. Furthermore, upon reduction, the axial ligand of the c heme of NiR-Pd changes from His17 to Met108. Finally, in the oxidized NiR-Pa structure, the N-terminal stretch of residues (1-29) of one monomer interacts with the other monomer (domain swapping), which does not occur in NiR-Pd. Here the structure of reduced NiR-Pa is described both in the unbound form and with the physiological product, NO, bound at the d1 heme active site. Although both structures are similar to that of reduced NiR-Pd, significant differences with respect to oxidized NiR-Pd were observed in two regions: (i) a loop in the c heme domain (residues 56-62) is shifted 6 A away and (ii) the hydroxide ion, which is the sixth coordination ligand of the heme, is removed upon reduction and NO binding and the Tyr10 side chain rotates away from the position adopted in the oxidized form. The conformational changes observed in NiR-Pa as the result of reduction are less extensive than those occurring in NiR-Pd. Starting with oxidized structures that differ in many respects, the two enzymes converge, yielding reduced conformations which are very similar to each other, which indicates that the conformational changes involved in catalysis are considerably diverse.

Crystallization↗
PubMed2006

Characterization of acrylic polyamide plastic embolization particles in vitro and in human tissue sections by light microscopy, infrared microspectroscopy and scanning electron microscopy with energy dispersive X-ray analysis.

Vascular embolization is a well-established practice for the treatment of tumors and vascular lesions. Rounded beads (microspheres) of various materials (collagen, dextran and trisacryl-polymer-gelatin) were developed to solve problems encountered with earlier versions of embolic material. We performed histochemistry, Fourier transform infrared microspectroscopy and scanning electron microscopy with energy dispersive X-ray analysis on two uterine and one hepatic specimen with unidentified intravascular foreign material, and examined a reference embolization product for comparison. The hematoxylin and eosin stained tissue sections showed multiple foci with unidentified intravascular foreign material and fibrous obliteration of vessel lumens. Only one case had a clinical history of previous embolization but without specifying the material used. One case was submitted for identification of a 'parasite'. The material stained positively with Sirius red and mucicarmine, variably with Masson's trichrome stain and Movat pentachrome, and did not stain centrally with periodic acid Schiff with diastase. Infrared spectrophotometric analysis of the material from all three cases demonstrated the spectrum of acrylic polyamide plastic. A control sample of EmboGold exhibited infrared microspectroscopic spectra similar to the three tissue specimens. Analysis by scanning electron microscopy with energy dispersive X-ray analysis demonstrated some differences in elemental composition between the tissue sections and the selected reference material. To our knowledge, this is the first report of infrared spectrophotometric analysis with scanning electron microscopy with energy dispersive X-ray analysis of an acrylic polyamide plastic embolization product both in vitro and in human histologic tissue sections. In cases lacking appropriate clinical information, identification by these methods and/or a panel of special stains may assist pathologists unfamiliar with this material's light microscopic appearance.

Acrylic Resins↗
PubMed2004

Identification of secondary metabolites in medicinal and spice plants by NIR-FT-Raman microspectroscopic mapping.

This paper demonstrates the special potential of vibrational NIR FT Raman microspectroscopy for the study of fennel fruits, chamomile inflorescence and curcuma roots to obtain detailed information about their microstructure and chemical composition. Microscopic Raman maps of fennel fruits demonstrate that anethole, which is the main essential oil component, is present in the whole mericarp with highest concentration at the top of the fruit. In situ measurements obtained of the essential oil cells are dominated by two bands observed at 1657 cm(-1) and 1609 cm(-1) which are characteristic for anethole. Raman images of chamomile inflorescence show that spiroethers, identified by significant bands between 2150 and 2250 cm(-1), are accumulated in the middle part of the flower head. Due to the intense curcumin bands in the Raman spectrum of curcuma root, the distribution of this dyeing substance can be clearly determined; highest concentration of curcumin was observed on the core of the root.

Flowering Tops↗
PubMed2004

A decade of vibrational micro-spectroscopy of human cells and tissue (1994-2004).

Instrumentation used in infrared microspectroscopy (IR-MSP) permits the acquisition of spectra from samples as small as 100 pg (10(-10) g), and as small as 1 pg for Raman microspectroscopy (RA-MSP). This, in turn, allows the acquisition of spectral data from objects as small as fractions of human cells, and of small regions of microtome tissue sections. Since vibrational spectroscopy is exquisitely sensitive to the biochemical composition of the sample, and variations therein, it is possible to monitor metabolic processes in tissue and cells, and to construct spectral maps based on thousands of IR spectra collected from pixels of tissue. These images, in turn, reveal information on tissue structure, distribution of cellular components, metabolic activity and state of health of cells and tissue.

Cell Physiological Phenomena↗
PubMed2005

Speciation of dissolved iron(II) and iron(III) in environmental water samples by gallic acid-modified nanometer-sized alumina micro-column separation and ICP-MS determination.

A method has been developed for the speciation of trace dissolved Fe(II) and Fe(III) in water by coupling gallic acid (GA) modified nanometer-sized alumina micro-column separation with inductively coupled plasma mass spectrometry (ICP-MS). The separation of Fe(II) and Fe(III) was achieved based on the obvious difference in reaction kinetics between Fe(II) and Fe(III) with GA. Fe(III) was selectively retained on the micro-column at pH 5.5-6.5, while Fe(II) could not be retained by the micro-column at the whole tested pH range of 1.0-6.5, and passed through the micro-column. The Fe(II) can be determined by ICP-MS directly without preconcentration/separation procedure, while Fe(III) retained on the micro-column was then eluted with 1.0 mL of 1 mol L(-1) HCl and determined by ICP-MS. The parameters affecting the separation of Fe(II) and Fe(III) were investigated systematically and the optimum separation conditions were established. Under the optimized conditions, the detection limits of 0.48 microg L(-1) and 0.24 microg L(-1) with relative standard deviation of 5.6% and 4.3%(C= 5 microg L(-1), n= 7) for Fe(II) and Fe(III) were found, respectively. No obvious effect on the speciation of Fe(II) and Fe(III) was found with the change of the ratio of Fe(II) and Fe(III) from 0 ratio 10 to 10 ratio 0. The proposed method was applied for the determination of trace Fe(II) and Fe(III) in environmental water and the recoveries for spiked samples were found to be in the range of 97-105%.

Fresh Water↗
PubMed2006

Towards biochemical reaction monitoring using FT-IR synchrotron radiation.

A lab-on-a-chip device made of CaF2 windows and SU-8 polymer was used for fluid lamination to achieve rapid mixing of two streamlines with a cross section of 300 x 5 microm each. Time resolved measurements of the induced chemical reaction was achieved by applying constant feeding low flow rates and by on-chip measurement at defined distances after the mixing point. Synchrotron IR microscopic detection was employed for direct and label-free monitoring of (bio)chemical reactions. Furthermore, using synchrotron IR microscopy the measurement spot could be reduced to the diffraction limit, thus maximizing time resolution in the experimental set-up under study. Based on computational fluid dynamic simulations the principle of the set-up is discussed. Experimental results on the basic hydrolysis of methyl chloroacetate proved the working principle of the experimental set-up. First results on the interaction between the antibiotic vancomycin and a tripeptide (Ac2KAA) involved in the build up of the membrane proteins of gram-positive bacteria are presented.

Animals↗
PubMed2000

Modeling lipid uptake in expanded polytetrafluoroethylene vascular prostheses and its effects on mechanical properties.

The radial transport across the wall of expanded polytetrafluoroethylene (ePTFE) arterial prostheses has a significant effect on lipid uptake observed in prostheses implanted in humans, which has been postulated to be one of the causes associated with implant failure. The goal of this study was to stimulate radial transport on a lipidic dispersion across the wall of an ePTFE prosthesis and investigate its effects on the circumferential mechanical properties of the prosthesis. An in vitro model was developed to simulate the lipidic radial transport across the wall. Lipids contained in a phosphatidylcholine dispersion were used as the transported molecules. Lipid concentration profiles were obtained after exposing commercial ePTFE prostheses to various transmural pressure and/or lipidic concentration gradients. Phospholipids gradually accumulated up to the external reinforcing wrap of the prosthesis, which clearly acted as a rigid barrier against lipid infiltration. Tensile tests performed on the virgin samples showed that the wrap was much more rigid than the microporous part of the prosthesis. After the lipid simulation, the rigidity of the wrap decreased with respect to what was observed for the virgin prosthesis. Finally, some clinical implications of this phenomena are discussed.

Biocompatible Materials↗
PubMed2003

Micro-lightguide spectrophotometry as an intraoral monitoring method in free vascular soft tissue flaps.

PURPOSE: The aim of this prospective study was to measure the hemoglobin oxygen saturation (HbO(2)%) and relative Hb concentration of free vascular soft tissue flaps using micro-lightguide spectrophotometry. The objective was to measure the normal range and topographic differences in HbO(2)% and rel. Hb conc. in tissue transfers before establishing this as a clinical method for monitoring perfusion and vitality. PATIENTS AND METHODS: In 39 patients who had received free vascular soft tissue flaps (34 radial forearm flaps; 8 latissimus flaps) to cover defects after tumor surgery, the capillary HbO(2)% in transferred tissue was measured spectrophotometrically preoperatively at the donor site and postoperatively up to the third postoperative day. On average about 500 hemoglobin spectra (200 to 800 spectra) were measured over each 24-hour period. Additionally, the relative Hb concentration was determined for the individual measuring times. The measurements were carried out topographically on the flap base, flap center, and flap periphery. RESULTS: The preoperative HbO(2) values at the donor site of free soft tissue flaps were between 20% and 40% in all topographic regions. In the case of clinically successful flaps, a normal distribution of the HbO(2) values of 20% to 80% was obtained in the immediate postoperative period, and from the second day on, a normal distribution of 45% to 60%. In the case of 2 flaps with partial necrosis, HbO(2) values of less than 10% to 15% were measured from the second postoperative day on. The relative Hb concentration had no influence on the amount of HbO(2)% measured in the transferred capillaries. In the postoperative phase, here was no topographic difference between the individual flap regions. CONCLUSIONS: As a noninvasive method, micro-lightguide spectrophotometry permits quantitative determination of HbO(2)% and relative Hb concentration over the entire surface of soft tissue flaps. In the case of partially unsuccessful flaps, HbO(2) values of less than 10% to 15% were measured beforehand, thus indicating that these HbO(2) values are not sufficient to support the vitality of the free tissue transfer. When combined with clinical observation, application can be recommended for the vitality measurement of free soft tissue flaps and permits more information to be obtained on topographic capillary perfusion conditions.

Adolescent↗