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PubMed1982

Vulvar intraepithelial neoplasia: correlation of nuclear DNA content and the presence of a human papilloma virus (HPV) structural antigen.

Immunoperoxidase localization of a human papilloma virus structural antigen (HPV) was attempted in 68 intraepithelial lesions of the vulva, 39 of which were analyzed for nuclear DNA content by microspectrophotometry. Overall, 5.9% (4/68) stained positive for HPV. Ninety percent (35/39) of the cases tested were aneuploid, and, of these, 2.8% (1/35) stained positive for HPV. In contrast, 50% (2/4) of the polyploid lesions were positive. Hence DNA microspectrophotometry and immunoperoxidase localization of HPV are useful coparameters for distinguishing wart virus infection (condylomata) from vulvar intraepithelial neoplasia. HPV is detected infrequently within aneuploid lesions, in keeping with the concept that epithelial maturation is required for virion assembly. Whether the HPV genome exists in a nonreplicative state within the aneuploid cell population is unknown.

Antigens, Viral↗
PubMed2004

Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals.

Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150-250 K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175 K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples.

Acetylcholinesterase↗
PubMedDate not supplied

A further study of dye batch variation in textile and carpet fibres.

Four sets of acrylic fibre samples were obtained from a company that dyes fabrics for the fashion industry. Between seven and ten different batches of fibres constituted each set. Comparison microscopy, visible and UV range microspectrophotometry and thin layer chromatography (TLC) were used to compare the dyes on each batch of fibres within the sets. Only one of the four sets exhibited variation within the batches. The differences were seen when both microscopical and analytical techniques were used. In addition, two further sets of samples had been obtained from a company that produces carpets for the car industry. The first set consisted of 26 batches of acid dyed orange nylon fibres. The second consisted of 21 batches of acid dyed mustard coloured nylon and direct dyed brown viscose fibres blended together. When the first set was viewed under UV light one batch had more pale orange fibres present and they fluoresced more brightly than the other fibres. This could be due to the blending with a different dye batch of fibre or due to poor dye uptake--the latter being more likely. When tested using visible and UV range microspectrophotometry and TLC, further dye batch variation was not detected. The second set was examined after separating the nylon and viscose fibres from each other. The nylon fibres were indistinguishable when a range of microscopical and analytical techniques were employed; however, the viscose fibres showed dye batch variation when TLC was used.

Journal Article↗
PubMedDate not supplied

The analysis and comparison of blue wool fibre populations found at random on clothing.

Fifty-eight garments were taped and searched for mid to dark blue wool fibres. These were then removed from the tapings, mounted on slides and examined using a high-power microscope (400x). A total of 2,740 blue wool fibres were identified and visible range microspectrophotometry (MSP) was performed on them. Three hundred independent blue wool populations were identified on 56 of the 58 garments searched. The lack of control fibres meant the spectral range of each population was unknown. The number of populations may have been underestimated by grouping together the fibres that had broad single peaks and a lack of distinguishing features in the spectra. Although blue wool is considered to be a common fibre type, 300 unique spectral shapes were identified by the use of microspectrophotometry alone. This demonstrates that the dyes used in the dyeing of blue wool are variable. Showing that many different populations of blue wool occur on a range of garments should ensure that the forensic scientist does not underestimate or understate the strength of evidence in cases where blue wool is found. Hopefully this work will enlighten scientists and enable them to also assess the true value of their findings when other commonly occurring fibres are encountered.

Journal Article↗
PubMedDate not supplied

The use of indigo derivatives to dye denim material.

Recently, attention was again drawn to the use of microspectrophotometry to distinguish between fibres dyed with indigo and seven derivative dyes. Under normal circumstances, fibres dyed with indigo (Vat Blue 1 or CI Number 73000) are not included in fibre-transfer examinations as, being ubiquitous, they do not usually have any evidential value. Indigo substitutes are used for fashion dyeing. If their use is widespread, the assumption that all denim material is dyed with Vat Blue 1 may need revising. A positive finding might make it necessary to reconsider the use of certain denim fabrics as capable of providing target fibres. Fibres from 160 samples of denim articles in current use were examined by microspectrophotometry to determine whether either indigo derivative dyes, or other dyes, had been used instead of the traditional indigo.

Journal Article↗
PubMed2003

Catching catalysis in the act: using single crystal kinetics to trap methylamine dehydrogenase reaction intermediates.

Methylamine dehydrogenase (MADH) is produced by a range of gram-negative methylotrophic and autotrophic bacteria, and allows the organisms to utilise methylamine as the sole source of carbon. The enzyme catalyses the oxidation of methylamine to formaldehyde and ammonia, leaving it in a two-electron reduced state. To complete the catalytic cycle, MADH is reoxidised via an electron transfer (ET) chain. The redox center in the enzyme is the organic cofactor tryptophan tryptophylquinone (TTQ) derived from the posttranslational modification of two Trp residues in the protein. This cofactor has spectral features in the visible region, which change during catalytic turnover, defining spectrally distinct reaction intermediates that reflect the electronic state of the TTQ. In the case of the Paracoccus denitrificans enzyme the physiologic ET chain involves the protein redox partner amicyanin (a blue copper protein). A stable binary (MADH/amicyanin) complex can be formed, and its crystal structure has been solved to 2.5 A resolution by Chen et al. [Biochemistry 21 (1992) 4959]. These crystals were shown to be competent for catalysis and ET by Merli et al. [J. Biol. Chem. 271 (1996) 9177] using single crystal polarised absorption spectroscopy. Through a novel combination of single crystal visible microspectrophotometry, X-ray crystallography and freeze-trapping, we have trapped reaction intermediates of the enzyme in complex with its physiological redox partner amicyanin in the crystalline state. We will present data confirming that catalysis and ET in the binary complex crystals can be tracked by single crystal visible microspectrophotometry. We will also show that the reaction pathway is unperturbed by the presence of cryoprotectant solution, enabling direct freeze-trapping of reaction intermediates within the crystal. We will present new data demonstrating that the binary complex crystals are also capable of exhibiting UV light-dependent oxidase activity, as observed in solution [Biochim. Biophys. Acta 1364 (1998) 297].

Bacterial Proteins↗
PubMed1977

The gecko visual pigments: a microspectrophotometric study.

1. A dual system of visual pigments having absorbance maxima in the green and blue respectively has been found in the retinas of geckos both by single cell microspectrophotometry and by the method of extraction. Microspectrophotometry has shown the system to be present in four species of geckos representing four genera. Along with previous work with extracts (Crescitelli, 1972) this indicates a fundamental property in this family of lizards. No other photopigments have been detected.2. For Gekko gekko, the species most intensively studied, the two pigments have absorbance maxima at 521 and 467 nm respectively. Both are based on vitamin A(1) and both are characterized by absorbance spectra that nearly conform to the Dartnall nomogram.3. In situ, the 521-pigment is not temperature-sensitive, and has virtually the same absorbance spectrum at 23 degrees C as at 5 degrees C. On extraction into digitonin solution, however, the absorbance spectrum of the pigment becomes temperature-dependent and, though identical when measured at low temperature (2-5 degrees C) with the constant in situ curve, is displaced to shorter wave-lengths at higher temperatures (10-25 degrees C). Thus the extract spectrum is relevant to the in situ spectrum only at low temperature (and in the presence of chloride ions).4. Unlike the rhodopsins of several vertebrates, the gecko 521-pigment displays no evidence of a meta-III stage in the sequence of products following photic bleaching, even at low temperatures. This is true for the pigment in situ as well as in the extracted condition.

Animals↗
PubMed1995

Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus.

The white rot fungi Ceriporiopsis subvermispora FP-90031-sp and Cyathus stercoreus ATCC 36910 were evaluated for their ability to delignify Bermuda grass (Cynodon dactylon) stems and improve biodegradability. Compositional and structural alterations in plant cell walls effected by the fungi were determined by nuclear magnetic resonance spectroscopy, gas chromatography of alkali-treated residues, microspectrophotometry, and electron microscopy. Contaminating bacteria and fungi, which grew from unsterilized Bermuda grass stems, did not alter the improvement in grass biodegradability by either of the fungi from that of gas-sterilized stems. The biodegradation of stems by ruminal microorganisms, after treatment for 6 weeks with C. subvermispora or C. stercoreus, was improved by 29 to 32% and by 63 to 77%, respectively; dry weight losses caused by pretreatment with the fungi were about 20% over that in untreated, control stems. Both fungi preferentially removed aromatics to carbohydrates, and C. subvermispora removed proportionately more guaiacyl units than did C. stercoreus. Substantial amounts of ester-linked p-coumaric and ferulic acids were removed by both fungi, and about 23 and 41% of total aromatics (determined after 4 M NaOH direct treatment) were removed from the plant biomass after incubation with C. subvermispora and C. stercoreus, respectively. UV absorption microspectrophotometry indicated that ester-linked phenolic acids were totally removed from the parenchyma cell walls, and these cells were readily and completely degraded by both fungi. However, aromatic constituents were only partially removed from the more recalcitrant sclerenchyma cell walls, resulting in variation in electron density and random digestion pits after incubation with fiber-degrading bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Basidiomycota↗
PubMed1975

Atomic absorption spectrophotometry applied to photographic densitometry.

For this study of photographic densitometry, sections of cartilage stained with Alcian Blue, safranin O and high iron diamine were photographed at x40 with Nikon photomicrography equipment on Kodak Panatomic X film with appropriate filters to enhance contrast. Portions of the developed negative films were selected from intercellular matrix regions, and circles of film equivalent in diameter to a 30-mu circle of tissue were obtained with a hand-held paper punch. Silver was eluted from the circles of film with 35% nitric acid, and the quantity of silver deposited on the film was determined by atomic absorption spectrophotometry as a measure of stain intensity. The intensity determined by this analytic procedure compared favorably with results obtained previously from the same tissue with microspectrophotometry. This method of silver analysis has advantages over earlier studies which used silver elution to determine photographic densitometry in its technical ease, accuracy and sensitivity. Furthermore, this method compares well with microspectrophotometry in its results and has the advantages of relative inexpensiveness and availability of equipment.

Animals↗
PubMed1996

Sodium polystyrene sulfonate (kayexalate) aspiration: histologic appearance and infrared microspectrophotometric analysis of two cases.

OBJECTIVE: Sodium polystyrene sulfonate (kayexalate) is a cation-exchange resin given enterally for the treatment of hyperkalemia. Aspiration of this material is a rare occurrence, but when visualized in the alveolus, it has a characteristic microscopic appearance that is virtually diagnostic. In two cases, recognition of the characteristic morphology of the foreign material raised the question of sodium polystyrene sulfonate. DESIGN: We used infrared spectroscopy to demonstrate the presence of this material in lung biopsies of two patients by identifying foreign body particles. Histories of the patients were reviewed for exposure to sodium polystyrene sulfonate. SETTING: Two lung specimens were referred to the Armed Forces Institute of Pathology with an unknown foreign material identified within the air spaces. PATIENTS: The lung biopsies were from two children, one postterm female infant who died at 3 days of life and a 4-year-old girl who underwent lung biopsy during surgical repair for tetralogy of Fallot. Both patients had received sodium polystyrene sulfonate previously for control of hyperkalemia. RESULTS: The lung specimens showed characteristic basophilic, amorphous foreign material in airspaces on histologic sections. The identity of this material was confirmed by Fourier transform infrared microspectrophotometry. CONCLUSIONS: To our knowledge, we report the first two cases of sodium polystyrene sulfonate aspiration in children. This material has a distinctive morphologic appearance on histologic sections, and its identity can be confirmed by Fourier transform infrared microspectrophotometry.

Cation Exchange Resins↗
PubMed1992

Photographic densitometry for quantitative DNA analysis of cytologic and histologic specimens.

We found that photographic densitometry (PD) is a useful technique for quantitative determinations of nuclear DNA content in clinical tumor material. Optimum conditions for the use of PD in clinical cytology and histopathology were worked out. A quantitative evaluation of the method was performed, particularly with respect to errors that may appear when measuring clinical tumor material. Our study showed that PD offers accurate DNA measurements in cytologic and histologic specimens. Ploidy level determinations in tumor cell populations in clinical material could be as accurately performed with PD as with scanning microspectrophotometry (SMP). Nuclear DNA content of individual cells as determined by PD correlated highly with nuclear DNA content determined by SMP (correlation coefficient, 0.96). Since the PD method is less influenced by background variation than are other image techniques (due to measurement of a photographic image), it is particularly useful in measurement of histopathologic sections, in which the background variation can introduce considerable errors. The method is also valuable with clinical cytologic smears, in which the presence of blood and other material disturbs the background. PD represents a valid complement to scanning microspectrophotometry and TV imaging systems, particularly for DNA analysis of tissue sections. Moreover, it can be applied easily in the clinical routine. Relevant tissue areas are selected and photographed by the pathologist or cytopathologist, and the measurement is performed by a laboratory technician.

Cell Nucleus↗
PubMed1996

[Histochemical study of human lens capsule--immunohistochemical analysis of collagen].

We studied the presence of different types of collagen (I, III, IV, V) in 31 human lens capsule specimens by use of enzyme labeled antibody (indirect method) and tried to quantify them by microspectrophotometry. We also quantified the total collagen content of lining layer and crust layer by microspectrophotometry in specimens stained by the van Gieson method. The presence of types I, III, IV and V collagen was demonstrated in the lens capsule and total collagen content in the lining layer was significantly richer than in the crust layer. Total collagen content tended to decrease with age over 60 and with the advance of lens opacity. Type V collagen increased more than type IV collagen after 60 years of age. These results suggest that the collagen in the lens capsule varies quantitatively and qualitatively with age and lens opacity.

Adolescent↗
PubMed1998

Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading.

INTRODUCTION: The articular cartilage collagen network and proteoglycans are subject to changes in deteriorating joint diseases. In this study, we exposed articular cartilage plugs to cyclic loading and investigated the properties of collagen network and proteoglycans in different zones of the articular cartilage. METHODS: Articular cartilage full-depth plugs were exposed in vitro to 4.1 MPa cyclic (0.5 Hz) loading for 1 to 20 hr and investigated using quantitative microscopic methods (i.e., polarized light microscopy, microspectrophotometry, and autoradiography). RESULTS: The loading caused packing or condensation of the tissue. In histological sections, the height of uncalcified articular cartilage decreased by an average of 12.8% (range, 4 to 19.7%). Loading increased the birefringence of collagen in the superficial cartilage (P < 0.05), with thickening of the zone up to 41.4% at 20 hr. The thickness of the intermediate zone increased also (22% at 1 hr and 434% at 20 hr). Concomitantly, the birefringence (P < 0.05) and the thickness of the deep zone decreased (18.5 to 27.8%). Loading for 4 hr increased the 35S-sulphate incorporation of the cartilage explants by an average of 67% (P < 0.05). The increase was most significant in the deep cartilage. A simultaneous increase was observed in the proteoglycan concentration of the cartilage; the staining intensity with safranin-O increased by 8.8% (P < 0.05). After 8 hr loading, this stimulation decreased; at 20 hr, loading caused a clear inhibitory effect on proteoglycan synthesis in the superficial zone. DISCUSSION: According to these results, the chosen loading regimen increased the thickness and collagen orientation in the superficial zone. In contrast, the thickness and birefringence in the deep cartilage were reduced. The proteoglycan metabolism of chondrocytes was first stimulated deep in the cartilage, but as the loading continued, the effect proved to be inhibitory (especially in the superficial part of uncalcified cartilage).

Animals↗
PubMed2002

Retinal characteristics of the ornate dragon lizard, Ctenophorus ornatus.

The retina of a diurnal insectivorous lizard, Ctenophorus ornatus (Agamidae) was investigated using microspectrophotometry and light and electron microscopy. A prominent broad yellow band was observed that extended across the mid-retina. The yellow coloration was found to originate from both oil droplets and diffuse pigmentation within cone inner segments. Microspectrophotometric analysis revealed yellow oil droplets with variable absorption of wavelengths below 520 nm and transparent oil droplets with no detectable absorptance between 350 and 750 nm. Cones with transparent oil droplets lacked the diffuse yellow pigmentation. The mean wavelengths of maximum absorbance of visual pigments in the isolated cone outer segments were at 440, 493, and 571 nm. The retina was found to possess a deep convexiclivate fovea located within the yellow band, slightly dorsotemporal of the retinal midpoint. The topography of the retinal ganglion cells revealed that the fovea was contained within an area centralis. Photoreceptors were either single (80%) or unequal double (20%) cones. Within the region of the fovea, the cones were approximately 20% the diameter of those in the peripheral retina. Colored oil droplets and yellow pigment may increase visual acuity by absorbing short wavelength light scattered either by the atmosphere or the optical structures of the eye. The presence of a fovea containing slender cone photoreceptors and three visual pigments suggests that the lizard has high acuity and the potential for color vision.

Animals↗
PubMed2003

Not all butterfly eyes are created equal: rhodopsin absorption spectra, molecular identification, and localization of ultraviolet-, blue-, and green-sensitive rhodopsin-encoding mRNAs in the retina of Vanessa cardui.

Surveys of spectral sensitivities, visual pigment spectra, and opsin gene sequences have indicated that all butterfly eyes contain ultraviolet-, blue-, and green-sensitive rhodopsins. Some species also contain a fourth or fifth type, related in amino acid sequence to green-sensitive insect rhodopsins, but red shifted in absorbance. By combining electron microscopy, epi-microspectrophotometry, and polymerase chain reaction cloning, we found that the compound eye of Vanessa cardui has the typical ultrastructural features of the butterfly retina but contains only the three common insect rhodopsins. We estimated lambda-max values and relative densities of the rhodopsins in the Vanessa retina (0.72, P530; 0.12, P470; and 0.15, P360) from microspectrophotometric measurements and calculations based on a computational model of reflectance spectra. We isolated three opsin-encoding cDNA fragments that were identified with P530, P470, and P360 by homology to the well-characterized insect rhodopsin families. The retinal mosaic was mapped by opsin mRNA in situ hybridization and found to contain three kinds of ommatidia with respect to their patterns of short wavelength rhodopsin expression. In some ommatidia, P360 or P470 was expressed in R1 and R2 opposed receptor cells; in others, one cell expressed P360, whereas its complement expressed P470. P530 was expressed in the other seven cells of all ommatidia. P470-expressing cells were abundant in the ventral retina but nearly absent dorsally. Our results indicated that there are major differences between the color vision systems of nymphalid and papilionid butterflies: the nymphalid Vanessa has a simpler, trichromatic, system than do the tetrachromatic papilionids that have been studied.

Amino Acid Sequence↗
PubMed2006

Photoreceptor layer of salmonid fishes: transformation and loss of single cones in juvenile fish.

The retinas of many vertebrates have cone photoreceptors that express multiple visual pigments. In many of these animals, including humans, the original cones to appear in the retina (which express UV or blue opsin) may change opsin types, giving rise to new spectral phenotypes. Here we used microspectrophotometry and in situ hybridization with cDNA probes to study the distribution of UV and blue cones in the retinas of four species of Pacific salmon (coho, chum, chinook, and pink salmon), in the Atlantic salmon, and in the rainbow/steelhead trout. In Pacific salmon and in the trout, all single cones express a UV opsin at hatching (lambda(max) of the visual pigment approximately 365 nm), and these cones later transform into blue cones by opsin changeover (lambda(max) of the blue visual pigment approximately 434 nm). Cones undergoing UV opsin downregulation exhibit either of two spectral absorbance profiles. The first is characterized by UV and blue absorbance peaks, with blue absorbance dominating the base of the outer segment. The second shows UV absorbance diminishing from the outer segment tip to the base, with no sign of blue absorbance. The first absorbance profile indicates a transformation from UV to blue phenotype by opsin changeover, while the second type suggests that the cone is undergoing apoptosis. These two events (transformation and loss of corner cones) are closely associated in time and progress from ventral to dorsal retina. Each double cone member contains green (lambda(max) approximately 510 nm) or red (lambda(max) approximately 565 nm) visual pigment (double cones are green/red pairs), and, like the rods (lambda(max) approximately 508 nm), do not exhibit opsin changeover. Unlike Pacific salmonids, the Atlantic salmon shows a mixture of UV and blue cones and a partial loss of corner cones at hatching. This study establishes the UV-to-blue cone transformation as a general feature of retinal growth in Pacific salmonids (genus Oncorhynchus).

Adaptation, Ocular↗
PubMed2001

Retinal photoreceptors and visual pigments in Boa constrictor imperator.

The photoreceptors of Boa constrictor, a boid snake of the subfamily Boinae, were examined with scanning electron microscopy and microspectrophotometry. The retina of B. constrictor is duplex but highly dominated by rods, cones comprising 11% of the photoreceptor population. The rather tightly packed rods have relatively long outer segments with proximal ends that are somewhat tapered. There are two morphologically distinct, single cones. The most common cone by far has a large inner segment and a relatively stout outer segment. The second cone, seen only infrequently, has a substantially smaller inner segment and a finer outer segment. The visual pigments of B. constrictor are virtually identical to those of the pythonine boid, Python regius. Three different visual pigments are present, all based on vitamin A(1.) The visual pigment of the rods has a wavelength of peak absorbance (lambda(max)) at 495 +/- 2 nm. The visual pigment of the more common, large cone has a lambda(max) at 549 +/- 1 nm. The small, rare cone contains a visual pigment with lambda(max) at 357 +/- 2 nm, providing the snake with sensitivity in the ultraviolet. We suggest that B. constrictor might employ UV sensitivity to locate conspecifics and/or to improve hunting efficiency. The data indicate that wavelength discrimination above 430 nm would not be possible without some input from the rods.

Animals↗
PubMed1993

Scotopic spectral sensitivity of the optomotor response in the green treefrog Hyla cinerea.

Amphibians are unusual among vertebrates in having two spectral classes of rod photoreceptors, unique amphibian "green" rods and typical vertebrate "red" rods. Although amphibians have been the subject of extensive research on visual function, it is not known whether possession of two classes of rods is a general feature of Amphibia, nor is it clear to what behaviors each class of rods contributes. The Hylidae comprise one of the largest families within Amphibia but have been little studied with respect to visual function. Here, we demonstrate the presence of green and red rods in Hyla cinerea by microspectrophotometry and provide evidence for the contribution of green rods to one visually based behavior, the optomotor response. In addition, we discuss the role of green and red rods in visually based behavior in light of apparently conflicting demands resulting from the need to maximize absolute sensitivity, visual acuity, and color sensitivity.

Animals↗