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PubMed2006

Comparison of maize brown-midrib isogenic lines by cellular UV-microspectrophotometry and comparative transcript profiling.

The molecular mechanisms underlying cell wall digestibility in maize (Zea mays L.) have been studied in three sets of maize brown-midrib isogenic lines in the genetic background of inbreds 1332 (1332 and 1332 bm3), 5361 (5361 and 5361 bm3), and F2 (F2, F2 bm1, F2 bm2, and F2 bm3). Two complementary approaches, SSH (suppression subtractive hybridization) and microarray-based expression profiling, were used to isolate and identify candidate genes in isogenic lines for bm mutants. Metabolic pathway analysis revealed that transcriptional events caused by altering the expression of a single bm gene involve all metabolic and signaling pathways. 53 ESTs were differentially expressed in all three isogenic bm3 comparisons, whereas 32 ESTs were consistently differentially expressed in different bm isogenic lines in F2 background. About 70% ESTs isolated by SSH were not present on the unigene microarray, demonstrating the usefulness of the SSH procedure to identify genes related to cell wall digestibility. Together with lignin analysis by cellular UV-microspectrophotometry, expression profiling in isogenic bm lines proved to be useful to understand alterations at the sub-cellular and molecular level with respect to lignin composition. The down-regulation of COMT affected the expression of CCoAOMT genes and caused a reduced content both of G and S units in bm3 mutants.

Base Sequence↗
PubMed1989

Demonstration of functional acetylcholinesterase on the soma of individual neurones of Aplysia by in vivo microspectrophotometry.

The presence of functional acetylcholinesterase is demonstrated in vivo on somatic membranes of single ganglionic neurones of Aplysia using concurrently microspectrophotometry and electrophysiology. The similarity of the effects of an irreversible blocker of acetylcholinesterase and of phospholipase C from Bacillus cereus suggests that acetylcholinesterase is anchored in the membrane via phosphatidylinositol.

Acetylcholinesterase↗
PubMed2004

Microspectrophotometry for structural enzymology.

For several decades, single-crystal microspectrophotometry has contributed to structural enzymology as a very useful complement to X-ray crystallography. In its most recent applications, it is the ideal tool to track chemistry as structure evolves in the course of time-resolved experiments, to identify freeze-trapped catalytic intermediates and to assess radiation-induced effects on enzyme crystals. To these goals, instruments have been developed to record optical spectra 'on-line' in the course of X-ray data collection, whereas more rigorous polarized absorption studies 'off-line' play an essential role in describing what protein function is retained in the crystalline state and correlating it with the observed structures.

Animals↗
PubMed1976

The automation of microspectrophotometry of tissue sections.

A system for quantitative microspectrophotometry with automatic data acquisition is described. It is relatively simple and less expensive than presently-available equipment. Fitted with the appropriate software, the system permits various types of probe selection, with both selection and measurement made under individual optimal conditions.

Computers↗
PubMed1992

Visual receptor cycle in normal and period mutant Drosophila: microspectrophotometry, electrophysiology, and ultrastructural morphometry.

Visual pigment, sensitivity, and rhabdomere size were measured throughout a 12-h light/12-h dark cycle in Drosophila. Visual pigment and sensitivity were measured during subsequent constant darkness [dark/dark (D/D)]. MSP (microspectrophotometry) and the ERG (electroretinogram) revealed a cycling of visual pigment and sensitivity, respectively. A visual pigment decrease of 40% was noted at 4 h after light onset that recovered 2-4 h later in white-eyed (otherwise wild-type, w per+) flies. The ERG sensitivity [in w per+ flies in light/dark (L/D)] decreased by 75% at 4 h after light onset, more than expected if mediated by visual pigment (MSP) changes alone. ERG sensitivity begins decreasing 8 h before light onset while decreases in visual pigment begin 2 h after light onset. These cycles continue in constant darkness (D/D), suggesting a circadian rhythm. White-eyed period (per) mutants show similar cycles of visual pigment level and sensitivity in L/D; per's alterations, if any on the D/D cycles were subtle. The cross-sectional areas of rhabdomeres in w per+ were measured using electron micrographic (EM) morphometry. Area changed little through the L/D cycle.

Animals↗
PubMed1969

Microspectrophotometry and the photoreceptor of Phycomyces I.

By applying microspectrophotometry to the sporangiophore of Phycomyces blakesleeanus wild-type and the albino car-10(-) type II, absorption spectra were obtained for 1- to 5-day cultures. Spectra in the growing-zone of the wild-type during Stage IVb, taken from 0.1 to 3 mm below the base of the sporangium, show two distinctly different spectra: one is more characteristic of a carotene, the other of a flavin. Combined, these absorption spectra reproduce closely the action spectrum. For the albino car-10(-), which is deficient in carotenes, only the spectrum characteristic of lumichrome or a reduced flavin was found. A c-type cytochrome was isolated from both strains which, if coupled with a flavin, could permit a photoreversible oxidation-reduction system. Birefringent crystals were observed to be aligned in the growing zone in which the photoreceptor is believed to lie. Micro-spectrophotometry of these crystals shows absorption peaks similar to those of riboflavin crystals.

Birefringence↗
PubMed1993

Visual pigment bleaching in isolated salamander retinal cones. Microspectrophotometry and light adaptation.

Visual pigment bleaching desensitizes rod photoreceptors greatly in excess of that due to loss of quantum catch. Whether this phenomenon also occurs in cone photoreceptors was investigated for isolated salamander red-sensitive cones. In parallel experiments, (a) visual pigment depletion by steps of bleaching light was measured by microspectrophotometry, and (b) flash sensitivity was measured by recording light-sensitive membrane current. In isolated cones, visual pigment bleaching permanently reduced flash sensitivity significantly below that due to the reduction in quantum catch, and there was little spontaneous recovery of visual pigment. The "extra" desensitization due to bleaching was most prominent up to bleaches of approximately 80% visual pigment and reached a level approximately 1 log unit beyond that due to loss of quantum catch. At higher bleaches, the effect of loss of quantum catch became more important. Bleaching did not greatly reduce the maximum light-suppressible membrane current. A 99% reduction of the visual pigment permanently reduced the circulating current by only 30%. Visual pigment bleaching speeded up the kinetics of dim flash responses. All electrical effects of bleaching were reversed on exposure to 11-cis retinal, which probably caused visual pigment regeneration. Light adaptation in photopic vision is known to involve significant visual pigment depletion. The present results indicate that cones operate with a maintained circulating current even after a large pigment depletion. It is shown how Weber/Fechner behavior may still be observed in photopic vision when the contributions of bleaching to adaptation are included.

Absorptiometry, Photon↗
PubMed1975

Microspectrophotometry of rhodopsin and metarhodopsin in the moth Galleria.

Fresh, frozen sections of the photoreceptor layer of the compound eye of the moth Galleria have been examined by microspectrophotometry, using 4 times 8 mum measuring beams that sampled from approximately two to four rhabdoms. The principal visual pigment absorbs maximally at 510 nm (P510), and on irradiation is converted to a thermally stable, pH-insensitive metarhodopsin with lambda max at 484 nm (M484) and a 43% increase in molar extinction coefficient. Subsequently, short wavelength irradiation of the metarhodopsin photoregenerates some P510, but the absence of an isosbestic point the cycle of spectral changes is consistent with the presence of smaller amounts of violet-or ultraviolet-sensitive visual pigment(s) that also are converted to a blue-absorbing metarhodopsin. Difference spectra for both P510 and M484 were measured, using hydroxylamine. The 484-nm metarhodopsin is reversibly converted to a form with lambda max at 363 nm by high concentrations of glycerol. Dark regeneration of rhodopsin in vivo after several minutes exposure of thoroughly dark-adapted animals to full sunlight requires several days.

Animals↗
PubMed1978

Correction of modal DNA values obtained by microspectrophotometry and tests for their shifts.

The technical problems in the application of statistical analyses of the nuclear DNA contents measured by plug or two-wavelength Feulgen microspectrophotometry were discussed. The skewness of the nuclear DNA content unimodally distributed in a cluster could be corrected by logarithmic transformation. A mean value of the logarithmically transformed DNA contents was a more logical characterization of cancer than a graphically obtained mode of nontransformed values. Equations were derived to estimate the number of cells in the G1- and S-phases of mitotic cycles contained within the cluster and to estimate the correction factor of the mean of transformed values of cells in the G1-phase. Thus statistical tests for a shift in the nuclear DNA content of the cells in question in the G1-phase from that of control cells were made possible.

Cell Cycle↗
PubMed2006

Color analysis of apparently achromatic automotive paints by visible microspectrophotometry.

Chromatic secondary pigments are utilized in achromatic automotive paints to create unique paint systems. These pigments may not be observable in reflected light; however, utilizing visible microspectrophotometry (MSP) discriminating data may be gathered. This study analyzed 160 apparently achromatic automotive paints via this technique for spectral evidence of secondary pigmentation. These results were compared with visual observations made via polarizing light microscopy. Positive spectral results were attained in approximately 25% of the black and gray/silver topcoat sample sets, whereas the white topcoat and gray undercoat set yielded no probative spectral data. The black sample set did yield several samples that produced spectral evidence of pigmentation when no visual chromatic data was observed. The results of this study suggest that paint analysis schemes should incorporate visible MSP for apparently achromatic black and gray/silver paint samples.

Journal Article↗
PubMed1985

Growth-activity related variation of a nuclear antigen in monolayer-culture cells quantitated by immunofluorometry and microspectrophotometry.

Antibodies of the hybridoma clone M 372/809 react with a nuclear antigen in growing cells. Using immunofluorometry and microspectrophotometry, the antigen was found to accumulate to a maximum coincident with the marked early increase in nuclear protein contents. The following DNA increase appeared when the intensity of the reaction with M 372/809 antibodies was decreasing. The antigen increase was inhibited by alpha-amanitin. It may therefore be associated with transcription early in the cell cycle.

Amanitins↗
PubMedDate not supplied

Feulgen microspectrophotometry of oral cancer and leukoplakia.

Feulgen microspectrophotometry was performed using the two wavelength method on 33 lesions and showed that five of ten carcinomas and 12 of 16 leukoplakias had diploid cell lines. This correlates well with similar findings in cervical cancer and dysplasia suggesting that changes in nuclear deoxyribonucleic acid content occur quite early in the evolution of cancer.

DNA↗
PubMed1985

Ultraviolet and blue light induced damage to the Drosophila retina: microspectrophotometry and electrophysiology.

Intense ultraviolet (UV) and blue stimulation decreases visual pigment concentration and increases long wavelength fluorescent emission in R1-6 photoreceptors in the white eyed fruit fly Drosophila melanogaster. We used microspectrophotometry to show that the threshold for visual pigment decrease is about 1 log unit lower for UV than for blue light (18.7 vs approximately 19.9 log quanta/cm2 respectively). UV and blue stimuli about 0.2 log units brighter had been shown to cause structural degeneration. Above the threshold for structural damage, visual pigment is decreased permanently while below this level, a recovery of visual pigment was achieved within several hours. Microspectrofluorometric data are partially consistent with the hypothesis that the visual pigment is converted into a fluorescent product which had been named M'. M' had been proposed to be a new form of metarhodopsin which absorbs chiefly in the yellow and which has a fluorescent emission in the red; long wavelength stimulation had been reported to regenerate the native visual pigment from M'. Our data suggest that the situation is significantly more complex than this simple model. For instance, we report that long wavelength stimulation regenerates only a small fraction of the visual pigment which had been decreased by UV or blue stimulation. Furthermore, several lines of evidence suggest that other fluorescent products are also created by intense UV and blue stimulation. We were particularly interested in the lower damage threshold for UV light because of the hypothesis that UV visual sensitivity is mediated by a sensitizing pigment which absorbs UV light and transfers its energy to the blue absorbing rhodopsin. Our data suggest that the UV light decreases the rhodopsin without preferentially decreasing the sensitizing pigment.

Animals↗
PubMed1987

The nuclear DNA content of vulvar skin using Feulgen microspectrophotometry and "four-quarter" analysis.

The nuclear DNA contents in subbasal (nonreplicating) cells of 161 vulvar biopsies from 31 patients were studied by Feulgen staining, integrated scanning microspectrophotometry (densitometry) and subsequent "four-quarter" analysis of the measurements. The results showed a constancy of the nuclear DNA content in the nonreplicating cells, regardless of the patient or the biopsy site.

Biopsy↗
PubMed1985

Some practical considerations in quantitative absorbance microspectrophotometry. Preparation techniques in DNA cytophotometry.

An experimental review of the Feulgen and gallocyanine-chrome-alum stains for quantitative cytophotometry of DNA in tissue sections yielded information on the preparation and staining of tissue for quantitative absorbance microspectrophotometry. (1) Tissues routinely fixed in formalin are suitable for either stain. Specimens fixed with glutaraldehyde-containing fixatives are not satisfactory for Feulgen staining, nor are ethanol-fixed specimens, unless they are post-fixed in formalin. (2) The pararosaniline dyes, used in the Feulgen stain, are sufficiently pure to use if a solution of the dye in ethanol shows an absorbance peak at 543 to 546 nm. (3) The Feulgen stain provides good reproducibility when the staining solution is adjusted to pH 1.5. (4) Gallocyanine is the best stain to use on Bouin-fixed or glutaraldehyde-fixed tissues. (5) Where fixation is an option, Carnoy and methanol-formalin-glacial acetic acid are excellent fixatives that can be followed by either stain. (6) Selection of the thickness of a tissue section involves a compromise. Requirements of minimum nuclear overlap and sharp focusing favor a section thickness of 4 micron to 6 micron. On the other hand, the requirement for full nuclear thickness, as judged by absorbance equivalent to that of a touch preparation, demands sections as thick as 8 micron in the case of mouse liver. Within this range, the optimum thickness, therefore, is determined by the particular tissue, the range of its nuclear sizes and its packing density. (7) The refractive index of the mounting medium should be closely matched to that of the background structure of the tissue sections. For animal tissues, we found that media with refractive indices of 1.54 to 1.56 are suitable.

Animals↗
PubMed1983

[Microspectrophotometry in the study of physiological and pathological events in erythrocytes].

Microspectrophotometry turns out to be the ideal method for an accurate study of single cells, namely of red blood cells, from the biochemical and physiological standpoint. Hereafter several possible applications are reported: a. the kinetics of gas-exchange (O2 and CO) in human erythrocytes has been made possible by the photosensitivity of the Hb-CO complex. In this way, it has been possible to establish that both ligands recombine with intraerythrocyte hemoglobin following a zero-order kinetic process. This suggested that diffusion of a ligand across an unstirred layer of buffer (approximately 5.10 mu thick) all around the cell could be the rate-limiting step of this recombination. Moreover, an intracellular facilitated oxygen diffusion has been observed and possible physiological implications have been shortly mentioned; b. the kinetics of intracellular polymerization of sickle cell hemoglobin in single red blood cells has been measured by quickly (approximately 1 msec) flashing-off the CO employing a cw Argon ion laser. The polymerization of deoxyhemoglobin has been followed by detecting the increased light scattering of the laser beam itself. The distribution of delay times (td) in several patients has been found significantly correlated to the severity of the disease, supporting the hypothesis that the td is a determinant factor in the pathophysiology of the disease. Possible therapeutic applications of this method are briefly discussed; c. the intraerythrocyte distribution of different hemoglobins of trout (Salmo irideus) among several erythrocytes has been observed by means of different physiochemical properties of single components. These turned out to be homogeneously present in all red blood cells studied.

Animals↗
PubMed1983

DNA cytometry of primary breast cancer. Comparison of microspectrophotometry and flow cytometry, and different preparation methods for flow cytometric measurements.

In 18 biopsy specimens from human breast carcinoma a comparison was made between DNA measurements obtained by microspectrophotometry (MSP) of Feulgen-stained nuclei in imprints and flow cytometry (FCM) of nuclei stained with ethidium bromide. For each specimen FCM was performed both with ethanol-fixed cells and unfixed cells. In addition, single cell suspensions were made from other 11 fresh mammary cancer biopsies. Parts of these suspensions were analysed both by MSP (Feulgen-stained smears) and FCM (ethanol-fixed, mitramycin-stained cells). The MSP histograms show selected tumour cells and tumour-like cells. This explains the higher proportion of cells with DNA content above the 2 c level. A good agreement was found between the results obtained by MSP and FCM with regard to the ploidy of the DNA stemline(s). FCM of fixed cells (multiple-step procedure) yielded a slightly lower proportion of diploid cells than FCM of the unfixed cells (one-step procedure), probably owing to loss of small cells during the different preparation steps. It is concluded that the results from DNA-histograms obtained from MSP and FCM can be compared as to DNA-stemline ploidy of the cell population but not as to the proportion of cells with non-diploid DNA-content.

Aneuploidy↗