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PubMed1996

The impact of fixatives on the binding of lectins to N-acetyl-glucosamine residues of human syncytiotrophoblast: a quantitative histochemical study.

We describe a quantitative histochemical method for demonstration of five N-acetyl-glucosamine binding lectins in the syncytiotrophoblast of human term placenta. The method employs biotinylated lectins and alkaline phosphatase-conjugated avidin. The alkaline phosphatase activity is detected by using 5-bromo-4-chloro-indoxyl phosphate as the substrate and nitroblue tetrazolium as the capture agent. The effect of 13 fixative solutions on specific lectin binding and nonspecific background staining was quantified by microspectrophotometry. Acid fixatives or fixatives containing mercuric chloride, e.g., Carnoy's and Zenker's fixatives, gave intense specific lectin binding and low background staining. Glutaraldehyde, carbodiimide, and ethanol resulted in low specific lectin binding and a very high background staining that was mainly due to endogenous placental alkaline phosphatase. Lectin binding to N-acetyl-galactosamine, mannose, galactose, and fucose was also significantly higher in sections from tissues fixed in an acid fixative compared with a neutral buffered fixative. Unfixed cryosections revealed a considerably lower degree of specific lectin binding compared with sections from fixed tissues. The activity of endogenous placental alkaline phosphatase was inhibited dose-dependently by mercuric chloride and decreased with L-phenylalanine concentration over the range of 7.8 x 10(-4) M to 5 x 10(-2) M, after which there was no further inhibition. Calf intestinal-type alkaline phosphatase conjugated to avidin was not inhibited by 5 x 10(-2) M L-phenylalanine. Endogenous placental biotin did not contribute significantly to background staining. Despite the high level of placental alkaline phsophatase, the intestinal-type alkaline phosphatase can be used as a marker enzyme in the sensitive ABC technique, provided that the nonspecific background is measured and substracted. Moreover, it is advisable to use an acid- and/or mercuric chloride-containing fixative and to add L-phenylalanine during incubation steps.

Acetylglucosamine↗
PubMed2003

17 Beta-estradiol suppresses AMPA-induced increases in regional cerebral O2 consumption.

We tested the hypothesis that 17 beta-estradiol would reduce the cerebral O2 consumption response resulting from glutamate receptor stimulation by alpha amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA). Fourteen ovariectomized rats were separated into 17 beta-estradiol (0.5 mg 21 day release pellet) and control (placebo pellet) groups to determine cerebral blood flow (14C-iodoantipyrine) and O2 consumption (microspectrophotometry). After topical cortical stimulation with 10(-3) M and 10(-4) M AMPA, cerebral blood flow increased significantly in both groups in a concentration-dependent manner. Cerebral O2 extraction was not significantly different in any region of the 17 beta-estradiol treated group. In the placebo treated group, the O2 extraction in the saline treated cortex and in the 10(-3) M AMPA treated cortex was significantly higher when compared to the 10(-4) M AMPA treated cortex. Cerebral O2 consumption in the control group increased by 20%, from 5.2 +/- 0.6 to 6.1 +/- 0.7, with 10(-4) M AMPA and significantly increased by 64% to 8.5 +/- 0.8 ml O2 min-1 100 g-1 with 10(-3) M AMPA. The 17 beta-estradiol group demonstrated no statistically significant difference in O2 consumption between the saline treated and AMPA treated cortex. Thus, 17 beta-estradiol reduced the effects of AMPA in increasing cerebral O2 consumption.

Animals↗
PubMed1994

Correlation of DNA content of laryngeal epithelial lesions to degree of malignancy.

PURPOSE: The aim of the present study was to determine the role of DNA content in laryngeal epithelial lesions of different degrees of malignancy. PATIENTS AND METHODS: A total of 53 lesions were studied, which included five benign tumors, 18 dysplasias, and 30 carcinomas of different degrees of differentiation and clinical stage. In all cases, the DNA content was determined by microspectrophotometry of paraffin-embedded Feulgen-stained biopsies. RESULTS: We found significant differences between the DNA-content histograms corresponding to each lesion group studied. Of special interest was the transition from moderate to severe dysplasia (characterized by a striking increase in the G2M-phase cells) and the transition from severe dysplasia to carcinoma (characterized by a brusque change in the modal ploidy of the lesion). CONCLUSION: The determination of DNA content, and study of DNA histogram, will help to assess degree of malignancy in epithelial lesions of the larynx.

Carcinoma↗
PubMed1999

Effect of AMPA receptor blockade on the control of cerebral O2 supply/consumption balance in newborn pigs.

Using 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466), we tested the hypothesis that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are important controllers of cerebral O2 supply/consumption balance in newborn piglets during both normoxia and hypoxia. Twenty-seven 2- to 7-day-old piglets were anesthetized with alpha-chloralose and were divided into four groups: 1) normoxia (n = 7), 2) GYKI 52466 (10 mg/kg, n = 7), 3) hypoxia (n = 6), and 4) hypoxia + GYKI 52466 (n = 7). We used [14C]iodoantipyrine to measure regional cerebral blood flow (rCBF) in mL/min/100 g, and we determined O2 extraction by microspectrophotometry, calculating cerebral O2 consumption (VO2) in mL O2/min/100 g in the cortex, hypothalamus, and pons. GYKI 52466 had no effect on regional VO2 or rCBF in normoxic piglets compared with controls. Hypoxia resulted in an increase in local VO2 and rCBF in the cortex and hypothalamus compared with controls: rCBF from 50 +/- 10 to 97 +/- 16 and VO2 from 2.4 +/- 0.5 to 3.7 +/- 0.4 in the cortex, and rCBF from 41 +/- 9 to 99 +/- 17 and VO2 from 2.5 +/- 1 to 3.8 +/- 0.5 in the hypothalamus. GYKI 52466 abolished this hypoxic flow effect in both the cortex (68 +/- 14) and hypothalamus (73 +/- 12). GYKI 52466 also blocked the increased VO2 in the cortex (2.5 +/- 0.4) and hypothalamus (3.0 +/- 0.5) of the hypoxic group. These findings suggest that the AMPA receptor is an important controller of VO2 in the cortex and hypothalamus during hypoxia in this immature porcine model.

Animals↗
PubMed1994

Effect of ketamine on heterogeneity of cerebral microregional venous O2 saturation in the rat.

Heterogeneity of microregional O2 supply and consumption balance exists in the brain. Previous studies have shown that anesthetics such as pentobarbital and isoflurane, decrease the heterogeneity of O2 saturation in the small veins of the brain. This study was performed to determine whether ketamine, an anesthetic, would alter this heterogeneity. Rats were anesthetized with isoflurane for the cannulation of the femoral artery and vein. Ninety minutes after discontinuation of isoflurane, they either received injections with ketamine 100 mg/kg intraperitoneally (IP) (n = 7) or were used as conscious controls (n = 7). In each rat, regional cerebral blood flow (rCBF) was determined by 14C-iodoantipyrine autoradiography, and microregional arterial and venous (diameter 20-100 microns) O2 saturation was measured by microspectrophotometry in the anterior cortex, posterior cortex, and pons. The average rCBF and O2 consumption were similar between the two groups in each brain region except in the anterior cortex, where the O2 consumption of the ketamine group was slightly higher than that of the conscious animals. The average regional O2 supply-to-consumption ratios were similar in the three brain regions in both of the groups. However, there was heterogeneity of the O2 saturation in small veins in all the brain regions that we studied. The coefficient of variation [CV = (SD/mean X 100] of venous O2 saturation of the ketamine group in the anterior cortex (19.8 +/- 7.2), in the posterior cortex (18.8 +/- 9.4), and in the pons (16.7 +/- 6.5) was not significantly different from that in the conscious animals (22.8 +/- 6.4, 23.1 +/- 5.3, and 15.7 +/- 6.4, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Proliferative behaviour of high-ploidy cells in two murine tumour lines.

The presence of high-ploidy cells in malignant tumours has long been documented. However, the biological significance of these cells is not known and there is a great deal of controversy over their proliferative potential. We have analysed the behaviour of these cells in two murine tumour lines, B16F10 melanoma and 3T3A31M angiosarcoma, determining their DNA content by microspectrophotometry and using time-lapse film studies. We have found a discrepancy between the presence of high-ploidy cells in metaphase and the absence of hyperploid telophases. High-ploidy metaphases may be aborted (mitotic polyploidization), prolonged in time or evolve in the form of multipolar, generally tripolar, mitoses. Our results suggest that high-ploidy cells are capable of proliferating, despite certain peculiarities in their cell cycle, and constitute a tumour subpopulation whose role in neoplasia merits further study.

Animals↗
PubMed1983

Microspectrophotometric and autoradiographic study of the timing and duration of pre-meiotic DNA synthesis in Paramecium caudatum.

Timing and duration of pre-meiotic G1, S and G2 phases in Paramecium caudatum have been clarified by microspectrophotometry of DNA content and autoradiography with [3H]thymidine ([3H]dThd) and [3H]deoxyuridinemonophosphate ([3H]dUMP). Microspectrophotometric measurement of DNA content showed that cells in stationary phase have a G1 micronucleus, and that pre-meiotic DNA synthesis begins with the micronuclear swelling and ends immediately before the nucleus enters meiotic prophase. The results indicate that the micronucleus is in G1 during the first 1.5 h after onset of mating reaction at 25 degrees C and in pre-meiotic S phase during the following 1 h, and that G2 phase is very short or lacking. Therefore, in P. caudatum, the transition point from pre-meiotic interphase to meiotic prophase is at or very close to the end of pre-meiotic DNA synthesis. Autoradiographic study showed that although [3H]dThd and [3H]dUMP were well utilized in pre-mitotic DNA synthesis, only [3H]dUMP was well utilized in pre-meiotic DNA synthesis. [3H]dThd was utilized very little in pre-meiotic DNA synthesis even if the precursor was taken into the cell, suggesting that the activity of thymidine kinase is low in these cells.

Animals↗
PubMed1988

Partition of protein (mass) to sister cell pairs at mitosis: a re-evaluation.

Since the 1960s it has been thought that there is to some extent a difference in the partition of mass to daughter cells at mitosis. Recent studies using modern techniques give further support to such a phenomenon, which has become almost an axiom in cell biology. It has been suggested that such unequal distribution of metabolic constituents at mitosis contributes to the dispersion in cell generation times. In the present work, PCC3 embryonal carcinoma (EC) cells were studied as undifferentiated G1 sister pairs by microspectrophotometry (MSP) following Feulgen-Naphthol Yellow staining (FNYS), in order to evaluate their protein content. Despite the considerable intraclonal intermitotic time heterogeneity found in undifferentiated PCC3 EC cells, it was concluded thta the postmitotic difference in mass (protein) between sister cell pairs exerts a minimal influence upon the cell population mass variability, whereas it was deduced to have an influence upon variation in interphase time duration when comparing sister cell pairs. This offers a cell-physiological explanation to the randomly distributed difference repeatedly found between sister cell generation times. Furthermore, there was no correlation seen between the mass difference found between sister cell pairs postmitotically and the size of the mother cell.

Animals↗
PubMed2005

Circadian rhythms of behavioral cone sensitivity and long wavelength opsin mRNA expression: a correlation study in zebrafish.

Using a behavioral assay based on visually mediated escape responses, we measured long-wavelength-sensitive red cone (LC) sensitivities in zebrafish. In a 24 h period, the zebrafish were least sensitive to red light in the early morning and most sensitive in the late afternoon. To investigate if the fluctuation of behavioral cone sensitivity correlates with opsin gene expression, we measured LC opsin mRNA expression at different times in the day and night under different lighting conditions. Under a normal light-dark cycle, the expression of LC opsin mRNA determined by real-time RT-PCR was low in the early morning and high in the late afternoon, similar to the fluctuation of behavioral cone sensitivity. This rhythm of LC opsin mRNA expression, however, dampened out gradually in constant conditions. After 24 h of constant light (LL), the expression of LC opsin mRNA dropped to levels similar to those determined in the early morning in control animals. By contrast, when the zebrafish were kept in constant dark (DD), the expression of LC opsin mRNA increased, to levels about 30-fold higher than the expression in the early morning in control animals. This day-night fluctuation in LC opsin mRNA expression was correlated to changes in opsin density in the outer segment of cone photoreceptor cells. Microspectrophotometry (MSP) measurements found significant differences in red cone outer segment optical density with a rhythm following the behavioral sensitivity. Furthermore, dopamine modulated the circadian rhythms in expression of LC opsin mRNA. Administration of dopamine increased LC opsin mRNA expression, but only in the early morning.

Animals↗
PubMed2006

Spectral sensitivity of the two-spotted goby Gobiusculus flavescens (Fabricius): a physiological and behavioural study.

Microspectrophotometry of Gobiusculus flavescens photoreceptors revealed a single rod visual pigment (lambdamaxat 508 nm) and the three cone pigments (lambdamax 456, 531 and 553 nm). The cone population was dominated by identical double cones containing the middle-wave-sensitive (MWS) pigment, but with a small number of non-identical MWS/LWS (long-wave-sensitive) and identical LWS double cones. Small populations of large single cones also contained either the MWS or LWS pigment. The short-wave-sensitive (SWS) pigment was found in small single cones. Lens transmission was great reduced below 410 nm. The spectral sensitivity of the behaviourally determined reaction distance (RD) to prey at a high irradiance level (0.5 micromol m-2 s-1) correlated with the maximum sensitivity of the MWS cones, both peaking around 530 nm. However, at a lower irradiance level (0.015 micromol m-2 s-1) such a correlation was not so apparent. The RD was greatly reduced, though still maintaining a peak around 530-550 nm, but with a relatively smaller reduction in RD at shorter wavelengths. Optomotor behaviour displayed a somewhat similar spectral sensitivity to the RD responses at the higher light intensity. However, the peak was at slightly longer wavelengths at 550 nm, suggesting a greater input from LWS cones to the optomotor response.

Animals↗
PubMed2006

Beauty in the eye of the beholder: the two blue opsins of lycaenid butterflies and the opsin gene-driven evolution of sexually dimorphic eyes.

Although previous investigations have shown that wing coloration is an important component of social signaling in butterflies, the contribution of opsin evolution to sexual wing color dichromatism and interspecific divergence remains largely unexplored. Here we report that the butterfly Lycaena rubidus has evolved sexually dimorphic eyes due to changes in the regulation of opsin expression patterns to match the contrasting life histories of males and females. The L. rubidus eye contains four visual pigments with peak sensitivities in the ultraviolet (UV; lambdamax=360 nm), blue (B; lambdamax=437 nm and 500 nm, respectively) and long (LW; lambdamax=568 nm) wavelength range. By combining in situ hybridization of cloned opsin-encoding cDNAs with epi-microspectrophotometry, we found that all four opsin mRNAs and visual pigments are expressed in the eyes in a sex-specific manner. The male dorsal eye, which contains only UV and B (lambdamax=437 nm) visual pigments, indeed expresses two short wavelength opsin mRNAs, UVRh and BRh1. The female dorsal eye, which also has the UV and B (lambdamax=437 nm) visual pigments, also contains the LW visual pigment, and likewise expresses UVRh, BRh1 and LWRh mRNAs. Unexpectedly, in the female dorsal eye, we also found BRh1 co-expressed with LWRh in the R3-8 photoreceptor cells. The ventral eye of both sexes, on the other hand, contains all four visual pigments and expresses all four opsin mRNAs in a non-overlapping fashion. Surprisingly, we found that the 500 nm visual pigment is encoded by a duplicate blue opsin gene, BRh2. Further, using molecular phylogenetic methods we trace this novel blue opsin gene to a duplication event at the base of the Polyommatine+Thecline+Lycaenine radiation. The blue opsin gene duplication may help explain the blueness of blue lycaenid butterflies.

Animal Communication↗
PubMed2006

Cone photoreceptor oil droplet pigmentation is affected by ambient light intensity.

The cone photoreceptors of many vertebrates contain spherical organelles called oil droplets. In birds, turtles, lizards and some lungfish the oil droplets are heavily pigmented and function to filter the spectrum of light incident upon the visual pigment within the outer segment. Pigmented oil droplets are beneficial for colour discrimination in bright light, but at lower light levels the reduction in sensitivity caused by the pigmentation increasingly outweighs the benefits generated by spectral tuning. Consequently, it is expected that species with pigmented oil droplets should modulate the density of pigment in response to ambient light intensity and thereby regulate the amount of light transmitted to the outer segment. In this study, microspectrophotometry was used to measure the absorption spectra of cone oil droplets in chickens (Gallus gallus domesticus) reared under bright (unfiltered) or dim (filtered) sunlight. Oil droplet pigmentation was found to be dependent on the intensity of the ambient light and the duration of exposure to the different lighting treatments. In adult chickens reared in bright light, the oil droplets of all cone types (except the violet-sensitive single cones, whose oil droplet is always non-pigmented) were more densely pigmented than those in chickens reared in dim light. Calculations show that the reduced levels of oil droplet pigmentation in chickens reared in dim light would increase the sensitivity and spectral bandwidth of the outer segment significantly. The density of pigmentation in the oil droplets presumably represents a trade-off between the need for good colour discrimination and absolute sensitivity. This might also explain why nocturnal animals, or those that underwent a nocturnal phase during their evolution, have evolved oil droplets with low pigment densities or no pigmentation or have lost their oil droplets altogether.

Aging↗
PubMed1994

STRUCTURAL BASIS FOR WAVELENGTH DISCRIMINATION IN THE BANKED RETINA OF THE FIREFLY SQUID WATASENIA SCINTILLANS

There is a greatly thickened region of retina in the ventral part of the eye of the firefly squid Watasenia scintillans, with an outer segment (OS) layer around 600 µm thick. The distal two-thirds of this OS layer is yellow and contains a visual pigment, based on 4-hydroxyretinal (A4), with an absorbance maximum at 470 nm. The proximal third is pink and contains a visual pigment, based on dehydroretinal (A2), with an absorbance maximum at 500 nm. Light and electron microscopic investigations demonstrate the presence of four types of photoreceptor cell. In the pink layer, three of the four types (alpha, ß and gamma) produce no microvilli and are columnar in structure. These cells form square microvillous rhabdoms only in the distal yellow layer. The fourth cell type (delta) produces microvilli in the pink layer only. These observations led us to propose that the A2-based visual pigment is contained in the pink-layer cells and that the A4-based visual pigment is contained in the three types of yellow-layer cells. The absorbance of fresh retina was determined by microspectrophotometry. The yellow OS layer, with an absorbance of 0.7 per 100 µm thickness at 470 nm, is expected to act as a short-wavelength cut-off filter to the underlying pink OS cells, shifting their photosensitivity peak by an estimated 50 nm to 550 nm. Possible wavelength discrimination by this squid is discussed.

Journal Article↗
PubMed1998

Photoreception in the planktonic larvae of two species of pullosquilla, a lysiosquilloid stomatopod crustacean

Optical microscopy, electron microscopy and microspectrophotometry were used to characterize pigments in the eyes of planktonic larvae of two species of the lysiosquilloid stomatopod Pullosquilla, P. litoralis and P. thomassini, which live sympatrically in French Polynesia. In contrast to the adult retina, which contains a diverse assortment of visual pigments in the main rhabdoms, the principal photoreceptors throughout the larval eyes of both species were found to contain a single rhodopsin with an absorption maximum ( max) close to 446 nm. The expression of this visual pigment may survive metamorphosis, since several adult rhodopsins occur at a similar spectral position. The retinas of these planktonic larvae also contain a novel yellow photostable pigment, which is arrayed in a regular pattern at the distal margin of the larval retina. The absorption spectrum of this pigment is well matched to the larval rhodopsin, suggesting that it acts to screen the rhabdoms from stray light. By replacing opaque, black screening pigment, the transparent yellow pigment may act together with a blue iridescent layer in the larval retina to reduce the visual contrast of the larval eye against downwelling and sidewelling light, while simultaneously acting as a retinal screen.

Journal Article↗
PubMed1999

The photoreceptors and visual pigments in the retina of a boid snake, the ball python (Python regius)

The photoreceptors and visual pigments of Python regius were studied using microspectrophotometry and scanning electron microscopy. The retina contains rods and cones, with rods constituting at least 90 % of the photoreceptor population. The rods are of a single type with long, narrow outer segments and are tightly packed. The wavelength of maximum absorbance ( &lgr; max) of the visual pigment in the rods is in the region of 494 nm. Two distinct types of cone are present. The most common cone, with a stout but stubby outer segment, contains a visual pigment with &lgr; max at approximately 551 nm. A relatively rare cone, with a long, slender outer segment, contains an ultraviolet-sensitive visual pigment with &lgr; max at approximately 360 nm. All the visual pigments have chromophores based on vitamin A1. The results are discussed in relation to the behavior of P. regius.

Journal Article↗
PubMed2001

Visual pigments in the early life stages of Pacific northwest marine fishes.

Microspectrophotometry was used to measure the visual pigments in the rods and cones of 22 species of marine fish larvae netted from the surface waters off Friday Harbor Laboratories, Washington, USA. 13 species had rods, 12 of which contained visual pigments with a wavelength of maximum absorbance near 500 nm, while one, the sand lance (Ammodytes hexapterus), had its absorbance maximum at 482 nm. The 22 species of fish larvae possessed varied combinations of single, double and twin cones, ranging in peak absorbance from 353 nm to 584 nm. Of these, green-sensitive single cones were present in 20 of the 22 species, and were the dominant cone type. Double and twin cones were present in 13 of the species. Most common were identical green-sensitive (twin) cones (in 11 species). Green/yellow-sensitive double cones occurred in four species. In a single instance (Hemilepidotus hemilepidotus) twin blue-sensitive, twin green-sensitive and double blue/yellow-sensitive cones were recorded. Of particular interest was the finding that 18 of the species had ultraviolet- and/or violet-absorbing single cones. It has been suggested that short-wavelength photosensitivity may be beneficial for planktivory by extending the spectral range over which vision can occur. The high percentage (82%) of ultraviolet and violet visual pigments in Pacific northwest fish larvae supports the prediction that short-wavelength sensitivity may be common in marine fish larvae.

Aging↗
PubMed2002

Developmental changes in the cone visual pigments of black bream Acanthopagrus butcheri.

The spectral absorption characteristics of the visual pigments in the photoreceptors of the black bream Acanthopagrus butcheri Munro (Sparidae, Teleostei), were measured using microspectrophotometry. A single cohort of fish aged 5-172 days post-hatch (dph), aquarium-reared adults and wild-caught juveniles were investigated. During the larval stage and in juveniles younger than 100 dph, two classes of visual pigment were found, with wavelengths of maximum absorbance (lambda(max)) at approximately 425 nm and 535 nm. Following double cone formation, from 40 dph onwards, the short wavelength-sensitive pigment was recorded in single cones and the longer wavelength-sensitive pigment in double cones. From 100 dph, a gradual shift in the lambda(max) towards longer wavelengths was observed in both cone types. By 160 dph, and in adults, all single cones had a lambda(max) at approximately 475 nm while the lambda(max) in double cones ranged from 545 to 575 nm. The relationships between the lambda(max) and the ratio of bandwidth:lambda(max), for changes in either chromophore or opsin, were modelled mathematically for the long-wavelength-sensitive visual pigments. Comparing our data with the models indicated that changes in lambda(max) were not mediated by a switch from an A(1) to A(2) chromophore, rather a change in opsin expression was most likely. The shifts in the lambda(max) of the visual pigments occur at a stage when the juvenile fish begin feeding in deeper, tanninstained estuarine waters, which transmit predominantly longer wavelengths, so the spectral sensitivity changes may represent an adaptation by the fish to the changing light environment.

Animals↗
PubMed2004

Differences in the optical properties of vertebrate photoreceptor classes leading to axial polarization sensitivity.

Polarization microspectrophotometry recordings were made to investigate possible differences in the way different spectral classes of photoreceptors from coho salmon (Oncorhynchus kisutch) absorb linearly polarized light. The results strongly suggest that rods and cones absorb transversely illuminating polarized light differently. Cones were found to exhibit a tilted optical geometry in which the maximum absorbance occurred when the E-vector was at a small angle to the transverse axis of the outer segment. Solutions to Maxwell's equations were deduced to investigate the effect of this tilt under conditions of axial illumination. Calculations show an approximate 10% difference in the absorbance of orthogonal polarizations, suggesting the possibility of axial dichroism in the cones of this species.

Journal Article↗