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PubMed2000

Protein diffusion in living skeletal muscle fibers: dependence on protein size, fiber type, and contraction.

Sarcoplasmic protein diffusion was studied under different conditions, using microinjection in combination with microspectrophotometry. Six globular proteins with molecular masses between 12 and 3700 kDa, with diameters from 3 to 30 nm, were used for the experiments. Proteins were injected into single, intact skeletal muscle fibers taken from either soleus or extensor digitorum longus (edl) muscle of adult rats. No correlation was found between sarcomere spacing and the sarcoplasmic diffusion coefficient (D) for all proteins studied. D of the smaller proteins cytochrome c (diameter 3.1 nm), myoglobin (diameter 3.5 nm), and hemoglobin (diameter 5.5 nm) amounted to only approximately 1/10 of their value in water and was not increased by auxotonic fiber contractions. D for cytochrome c and myoglobin was significantly higher in fibers from edl (mainly type II fibers) compared to fibers from soleus (mainly type I fibers). Measurements of D for myoglobin at 37 degrees C in addition to 22 degrees C led to a Q(10) of 1.46 for this temperature range. For the larger proteins catalase (diameter 10.5 nm) and ferritin (diameter 12.2 nm), a decrease in D to approximately 1/20 and approximately 1/50 of that in water was observed, whereas no diffusive flux at all of earthworm hemoglobin (diameter 30 nm) along the fiber axis could be detected. We conclude that 1) sarcoplasmic protein diffusion is strongly impaired by the presence of the myofilamental lattice, which also gives rise to differences in diffusivity between different fiber types; 2) contractions do not cause significant convection in sarcoplasm and do not lead to increased diffusional transport; and 3) in addition to the steric hindrance that slows down the diffusion of smaller proteins, diffusion of large proteins is further hindered when their dimensions approach the interfilament distances. This molecular sieve property progressively reduces intracellular diffusion of proteins when the molecular diameter increases to more than approximately 10 nm.

Animals↗

PubMed1978

Single cell observations of gas reactions and shape changes in normal and sickling erythrocytes.

Microspectrophotometry has been applied to single red blood cells to reinvestigate the linked processes of diffusion of gases inside the erythrocyte and their combination with hemoglobin. The experiments took advantage of the photosensitivity of the cabron monoxide derivative of hemoglobin, which allows ligand release from the CO-saturated red cells under strong illumination and recombination when the light is switched off. The photochemical method was also used to study the kinetics of sickling on ligand removal in single erythrocytes of Hb S carriers. The results give new information on the mechanism of the sickling process.

Anemia, Sickle Cell↗

PubMed1984

A barrier to lateral diffusion of porphyropsin in Necturus rod outer segment disks.

Microspectrophotometry was used to study lateral diffusion of the visual pigment, porphyropsin , in the disk membrane in intact mudpuppy (Necturus maculosus) rod outer segments (ROS), isolated in frog Ringer's solution. A concentration gradient of unbleached visual pigment was produced on the disks by rapidly photobleaching 40% of the pigment in an area spanning 1/4 or 1/2 of the cell's width. The change in optical density of the cells at 580 nm was then followed with time on either the bleached or unbleached side. The temperature dependence of porphyropsin diffusion yielded a Q10 of 2.5 between 10 and 20 degrees C with an activation energy of 12 +/- 2 kcal. At completion of pigment diffusion, the center and edge of the disk had, respectively, attained only 90 and 55% of the concentration expected. Computed diffusion coefficients (5.4 X 10(-9) cm2/s) were similar at the center and periphery of the disk immediately after the flash, however, an additional slow component for diffusion was detected at the periphery. A comparison of optical density at 525 nm along the diameter of ROS before and after the flash showed a persistent (20 min) postbleach concentration gradient of unbleached porphyropsin . This suggests that 15% of the prophyropsins may be sequestered into distinct areas on a mudpuppy disk and are not free to diffuse over the whole surface. This argument is supported by the observation that mudpuppy disks are separated into petal -shaped regions by incisures, some of which penetrate nearly to the disk center.

Animals↗

PubMed2002

17Beta-estradiol blocks NMDA-induced increases in regional cerebral O(2) consumption.

We tested the hypothesis that 17beta-estradiol would reduce the cerebral O(2) consumption response to stimulation of N-methyl-D-aspartate (NMDA) receptors. We determined NMDA receptor density in 10 ovariectomized Wistar female rats equally divided into a control group and 17beta-estradiol (500 microg/21 days) treated group. An autoradiographic assay using 125I-MK-801, an NMDA antagonist, was used to measure specific binding to NMDA receptors. Another 14 ovariectomized rats were separated into 17beta-estradiol and control groups to determine cerebral blood flow (14C-iodoantipyrine) and O(2) consumption (microspectrophotometry). 17Beta-estradiol caused a 20% decrease in specific binding to cortical NMDA receptors. After topical cortical stimulation with 10(-3)M and 10(-4)M NMDA, blood flow increased significantly in control from 73+/-5 in the saline treated cortex to 110+/-8 ml/min/100 g with 10(-3)M NMDA. In contrast, there was no significant change in blood flow in the 17beta-estradiol treated animals. Cerebral O(2) extraction increased significantly in the 10(-3)M NMDA treated cortex in both groups. Cerebral O(2) consumption in the control group significantly increased by 53%, from 3.7+/-0.2 to 5.7+/-0.5 with 10(-4)M NMDA and 72% to 6.4+/-2.4 ml O(2)/min/100 g with 10(-3)M NMDA. The 17beta-estradiol group demonstrated no significant difference between the saline treated and NMDA treated cortex. Thus, 17beta-estradiol blocked the effects of NMDA on cerebral O(2) consumption and this was associated with a slightly decreased number of NMDA receptors.

Animals↗

PubMed1997

Reduced myocardial cyclic GMP increases myocardial O2 consumption in control but not renal hypertension-induced cardiac hypertrophy.

OBJECTIVES: We tested the hypothesis that a reduction in myocardial cyclic GMP would increase myocardial O2 consumption and that renal hypertension (One Kidney-One Clip, 1K1C)-induced cardiac hypertrophy would change this relationship. METHODS: Either vehicle or LY83583 (10(-3) M, a guanylate cyclase inhibitor) was topically applied to the left ventricular surface of control of 1K1C anesthetized open-chest New Zealand white rabbits (N = 38). Coronary blood flow (radioactive microspheres) and O2 extraction (microspectrophotometry) were used to determine subepicardial (EPI) and subendocardial (ENDO) O2 consumption and myocardial cyclic GMP was determined by radioimmunoassay. RESULTS: The heart weight/body weight ratio was greater in the 1K1C rabbits (3.16 +/- 0.20) than controls (2.58 +/- 0.08 g/kg). Systolic blood pressure was higher in 1K1C rabbits (116 +/- 8 mm Hg) than controls (80 +/- 6), but topical LY83583 had no significant hemodynamic effects. LY83583 significantly and similarly decreased EPI cyclic GMP in both control (7.9 +/- 1.2 to 6.0 +/- 1.0 pmol/g) and 1K1C (7.7 +/- 1.2 to 5.3 +/- 0.9) hearts and control ENDO (8.7 +/- 1.7 to 7.2 +/- 1.2) but not 1K1C ENDO (6.7 +/- 0.5 to 5.7 +/- 1.1). Myocardial O2 consumption was significantly increased in control with LY83583 (EPI 6.6 +/- 1.1 to 15.6 +/- 1.4 and ENDO 7.2 +/- 0.9 to 14.2 +/- 0.7 ml O2/min/100 g), but not in 1K1C hearts (EPI 12.1 +/- 1.0 to 12.9 +/- 1.2 or ENDO 11.4 +/- 0.7 to 12.9 +/- 0.9). CONCLUSIONS: Thus myocardial O2 consumption was only increased by LY83583 in control hearts, but LY83583 decreased cyclic GMP similarly in both the control and 1K1C EPI. This indicated, at least in the EPI, a dissociation of the inverse relationship between the myocardial level of cyclic GMP and O2 consumption in the 1K1C rabbit heart.

Aminoquinolines↗

PubMed1998

On derivation of spectral sensitivities of the human cones from trichromatic colour matching functions.

Despite the recent advance made by using the direct methods of retinal densitometry, microspectrophotometry and suction electrophysiology, the psychophysical approach based on colour matching data still remains an important source of accurate information about the spectral sensitivity of the cone photoreceptors in the human visual system. However, the commonly used technique of estimating cone sensitivities, based on the assumption that dichromacy is caused by the lack of one of the three types of the cone photoreceptors, requires the colour matching functions not only from trichromatic observers but from dichromats as well. Here we evaluate an alternative approach, originally put forward by Bongard and Smimov, that derives cone spectral sensitivities from colour matching functions only; without resorting to colour deficiency or any other data. When applied to CIE standard colour matching functions, this method yields curves of spectral sensitivities that are close to the classical Smith-Pokorny fundamentals, though the long-wave cone is shifted towards the short-wave region of the spectrum by 5 nm, as compared with Smith and Pokorny's results.

Color Perception↗

PubMed1982

Nuclear feulgen DNA content and nuclear size in human breast carcinoma.

Biopsy specimens from 29 patients with operable breast carcinomas were examined by Feulgen-DNA microspectrophotometry. A diploid DNA stemline was found in ten tumors, seven of which were stage I carcinomas. In 19 tumors, ten of which were stage II cancers, a non-diploid DNA stemline was observed. The diploid tumors were most often estrogen receptor-positive (nine of nine examined carcinomas), whereas 13 of 19 non-diploid tumors studied did not contain estrogen receptors. The mean nuclear size of the non-diploid tumor cells was increased compared with that of the diploid malignant cells. Three grade I carcinomas were diploid, whereas three grade III tumors were non-diploid. Of the 23 grade II carcinomas, seven were diploid and 16 non-diploid. Hum Pathol 13:626-630, 1982.

Adenofibroma↗

PubMed2001

Special cytochemistry in cell biology.

Cytochemistry is a science of localizing chemical components of cells and organelles on histological sections by using various techniques. We first aimed at studying cytochemistry by developing new techniques using various principles such as enzyme cytochemistry, microincineration, microspectrophotometry, radioautography, cryo-techniques, X-ray microanalysis and immunocytochemistry. We first concentrated on developing methodologies in the 1960s to 1970s. We then applied these special techniques to various kinds of cells in men and animals. Earlier, I proposed to classify these methods into three categories, i.e., chemical, physical, and biological techniques. The methodology has been well developed to form a new science which should be designated as "general cytochemistry" similarly to the general histology. On the other hand, these techniques should be applied to various cells in various organ systems, such as the skeletal, muscular, digestive, respiratory, urinary, reproductive, endocrine, circulatory, nervous and sensory systems similarly to the special histology or the histology of organs. I summarize the results of cytochemical studies on cells of various organs carried out in our laboratory during these 44 years since 1955. The results obtained from cytochemical studies applied to various cells in respective organ systems should be designated as "special cytochemistry."

Aging↗

PubMed1998

Observations on pathology trends in 62,537 prostate biopsies obtained from urology private practices in the United States.

OBJECTIVES: To evaluate pathology trends of 62,537 first-time prostate needle-core biopsies submitted by office-based urologists, processed at a single pathology laboratory. METHODS: Prostate biopsy cases obtained over a 2-year period were assessed. Patient information included age, digital rectal examination (DRE) status, and prostate-specific antigen (PSA) serum levels. Biopsy pathology results included the number of tissue samples per case, Gleason score, presence of Gleason grades 4 or 5, percent of biopsy length with evidence of cancer, number of samples with cancer per biopsy, and determination of DNA ploidy status using microspectrophotometry. RESULTS: Adenocarcinoma, suspicious lesions, and isolated high-grade prostatic intraepithelial neoplasia (PIN) were diagnosed in 38.3%, 2.9%, and 4.1% of the biopsies, respectively. For each serum PSA and age range assessed, the positive biopsy rate and incidence of critical pathologic features increased consistently. The average percentage of biopsy length with evidence of tumor, the percentage of cases with Gleason grades 4 or 5, and the percentage of cases with an abnormal DNA ploidy all decreased significantly over the 2-year period (P <0.01). CONCLUSIONS: The number of tissue cores and anatomic sites (locations) being sampled per biopsy are increasing. The tumor size detected and percentage of cases with Gleason grades 4 and 5 are decreasing. There has been a slight increase in the number of biopsies performed on men younger than 60 years of age and a slight decrease in biopsies performed on men older than 70 years of age. The decline in meaningful pathologic features observed in biopsies over time may be clinically relevant to improved disease management.

Aged↗

PubMed2000

Microbial analysis at the single-cell level: tasks and techniques.

The heterogeneity of microorganisms themselves is orders of magnitude greater than the heterogeneity of perspectives from which they are contemplated by human observers. Even closely related species may exhibit marked differences in biochemistry and behavior, and, under many conditions, similar, striking heterogeneity may exist within a clonal population of organisms which, in the aggregate, occupy too small a region of space to be visible to the unaided human eye. Using methods of microscopy, microspectrophotometry, and cytometry developed and refined since the 1960s, it is now possible to characterize the physiology and pharmacology of individual microorganisms, and, in many cases, to isolate organisms with selected characteristics for culture and/or further analysis. These methods include fluorescent and confocal microscopy, scanning and image cytometry, and flow cytometry. Fluorescence measurements are particularly important in single-cell analysis; they allow demonstration and quantification of cells' nucleic acid content and sequence, of the presence of specific antigens, and of physiologic characteristics such as enzyme activity and membrane potential. Multiparameter cytometry, combined with cell sorting, provides insight into population heterogeneity and allows selected cells to be separated for further analysis and culture. The technology is applicable to a wide range of problems in contemporary microbiology, including strain selection and the development of antimicrobial agents.

Bacteria↗

PubMed1997

Structural analysis of compound I in hemoproteins: study on Proteus mirabilis catalase.

Ferryl catalysis has attracted considerable interest, because a diverse variety of enzymes use ferryl intermediates to perform difficult chemistry. The structure of the reactional intermediate compound I of Proteus mirabilis catalase (PMC) has been solved using time-resolved X-ray diffraction techniques and single crystal microspectrophotometry. Formation of compound I is characterized by significant changes in the absorbance spectrum, and the creation of an oxoferryl group on the distal side of the heme. This group is clearly visible in the X-ray electron density maps. An unidentified electron density, likely to be an anion because of the nature of its environment, appears during the reaction, in a site distant from the heme. The structure of compound I in PMC is compared with that of compound I in cytochrome c peroxidase (CCP).

Catalase↗

PubMed1980

[Diagnosis of stromal hyperplasia of the endometrium (author's transl)].

In three cases of endometrial stromal hyperplasia, nuclear DNA-content was determined by Feulgen microspectrophotometry. In two cases DNA-content exhibited the diploid pattern. In the third case values ranged from diploid to tetraploid, also showing an additional peak for hypotetraploid values. The two cases with diploid DNA patterns are considered to have less malignant potential. This corresponds well to clinical follow-up examinations performed three and nine years later, where no progression to malignant growth was established. The pronounced proliferative tendency observed in the third case would make progression to malignancy appear highly probable.

Aged↗

PubMed1986

Assessment of validity of DNA measurement in tissue sections by model simulation.

Computer modelling is used to simulate nuclear segments obtained by random sectioning through tissue. A few more computations lead to DNA measurement simulation. Two methods of DNA measurement (a direct one and a variant of the "plug" method) are tested on simulated diploid, tetraploid and octaploid cell populations. The two methods result in negatively skewed DNA frequency distributions. Both the right skewness and the coefficient of variation of measurements are increasing with the ploidy level because nuclear DNA content is assumed to be related to nuclear size in the chosen model. Observed mean values are biased underestimates of expected values but are strongly correlated to the degree of ploidy. The variant of the "plug" method gives rise to smaller coefficients of variation. Finally, the bias introduced by measuring nuclear segments instead of whole nuclei increases the variance of measurements but contributes to less than half the experimentally observed variance. Our conclusion is that microspectrophotometry on tissue sections is a valuable method for DNA content evaluation of small clusters of pathological cells as one may find in endoscopic biopsies.

Cell Nucleus↗

PubMed2003

Inter-comparison of unrelated fiber evidence.

The foreign textile fibers recovered from one item of evidence from each of 20 unrelated crimes in three categories (bank robbery, kidnapping, and homicide) were cross-compared. The items of evidence were scraped to remove the trace evidence and a sample of the collected fibers was examined using a standard scheme of analysis. The fibers were examined with light microscopy (including polarized light microscopy), fluorescence microscopy, and microspectrophotometry. The fibers were divided into natural and manufactured groups and then categorized by color and generic (polymer) class. Cross-comparing all 2083 fibers resulted in 2,168,403 comparisons, after removing duplicate (same fiber) comparisons. Colorless and denim fibers were excluded from this study. No two fibers were found to exhibit the same microscopic characteristics and analytical properties. Therefore, it is rare to find two unrelated items that have foreign fibers that are analytically indistinguishable. These results corroborate other population studies conducted in Europe and target fiber studies conducted both in the US and in Europe.

Journal Article↗

PubMed2001

The effect of tibial lengthening using the Ilizarov method on the cartilage and the menisci of the knee joint.

In order to investigate possible acute damage to the knee joint cartilage and the menisci during tibial lengthening, sixteen young beagle dogs underwent 30% lengthening of the right tibia of 2.5 cm by callus distraction at a distraction rate of twice 0.5 mm per day. A further four dogs comprised the control group with fixator and osteotomy but without lengthening. After a distraction period of 25 days half the dogs were killed (group A) while the other half (eight dogs with limb lengthening and two dogs without) were killed after a further period of 25 days (group B). At the end of the study, the menisci were removed together with three cartilage-bone cylinders from both femoral condyles from the weight-bearing zones as well as from the corresponding tibial condyles. Serial sections from the menisci were stained with haematoxylin and eosin (H&E) and Elastica van Gieson. Sections of the cartilage-bone cylinders were stained with H&E and safranin-O. Cartilage thickness was measured and the glycosaminoglycan content of the joint cartilage was determined using microspectrophotometry. None of the histological preparations obtained from the untreated and distracted sides showed any signs of damage to the cartilage or to the menisci. There were no significant differences between cartilage thickness and proteoglycan content of the untreated side and the lengthened side. Thus, tibial lengthening using the llizarov method does not appear to cause acute damage to the cartilage of the knee joint or to the menisci.

Animals↗

PubMed1994

Laue diffraction study on the structure of cytochrome c peroxidase compound I.

BACKGROUND: Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide. This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group. RESULTS: Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell. The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour. The structure of the intermediate was determined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures. CONCLUSIONS: The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment.

Amino Acid Sequence↗

PubMed2001

Vertebrate photoreceptors.

The basis of the duplex theory of vision is examined in view of the dazzling array of data on visual pigment sequences and the pigments they form, on the microspectrophotometry measurements of single photoreceptor cells, on the kinds of photoreceptor cascade enzymes, and on the electrophysiological properties of photoreceptors. The implications of the existence of five distinct visual pigment families are explored, especially with regard to what pigments are in what types of photoreceptors, if there are different phototransduction enzymes associated with different types of photoreceptors, and if there are electrophysiological differences between different types of cones.

Adaptation, Ocular↗

PubMed1998

The eyes of deep-sea fish. I: Lens pigmentation, tapeta and visual pigments.

Deep-sea fish, defined as those living below 200 m, inhabit a most unusual photic environment, being exposed to two sources of visible radiation; very dim downwelling sunlight and bioluminescence, both of which are, in most cases, maximal at wavelengths around 450-500 nm. This paper summarises the reflective properties of the ocular tapeta often found in these animals, the pigmentation of their lenses and the absorption characteristics of their visual pigments. Deep-sea tapeta usually appear blue to the human observer, reflecting mainly shortwave radiation. However, reflection in other parts of the spectrum is not uncommon and uneven tapetal distribution across the retina is widespread. Perhaps surprisingly, given the fact that they live in a photon limited environment, the lenses of some deep-sea teleosts are bright yellow, absorbing much of the shortwave part of the spectrum. Such lenses contain a variety of biochemically distinct pigments which most likely serve to enhance the visibility of bioluminescent signals. Of the 195 different visual pigments characterised by either detergent extract or microspectrophotometry in the retinae of deep-sea fishes, ca. 87% have peak absorbances within the range 468-494 nm. Modelling shows that this is most likely an adaptation for the detection of bioluminescence. Around 13% of deep-sea fish have retinae containing more than one visual pigment. Of these, we highlight three genera of stomiid dragonfishes, which uniquely produce far red bioluminescence from suborbital photophores. Using a combination of longwave-shifted visual pigments and in one species (Malacosteus niger) a chlorophyll-related photosensitizer, these fish have evolved extreme red sensitivity enabling them to see their own bioluminescence and giving them a private spectral waveband invisible to other inhabitants of the deep-ocean.

Animals↗