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PubMed1984

Comparative Studies of Fluorescence from Mesophyll and Guard Cell Chloroplasts in Saxifraga cernua: Analysis of Fluorescence Kinetics as a Function of Excitation Intensity.

The chlorophyll fluorescence induction curves from mesophyll and guard cell chloroplasts of Saxifraga cernua, including both the fast (O to P, the transients involved in the rise in variable fluorescence) and slow (P to steady state fluorescence due to quenching) components, were characterized over a range of excitation intensities using microspectrophotometry (with epi-lumination) equipped with apertures designed to eliminate cross contamination of the fluorescence signal between the two chloroplast types. At low excitation intensities, the fast fluorescence kinetics from guard cell plastids showed an extended I to D phase and a more rapid appearance of P while minimal quenching from P to steady state fluorescence was observed compared to the transients from mesophyll chloroplasts suggesting a lower activity of photochemical (electron movement via carriers between donor and acceptor sites) and nonphotochemical (such as membrane conformational changes) events which regulate the fluorescence induction curve kinetics. As the excitation intensity was increased, the quenching rates of guard cells were faster at initiating conditions for photophosphorylation and the fast and slow fluorescence kinetics from guard cells resembled those of the mesophyll cells.Guard cell chloroplasts of S. cernua from intact epidermal peels showed a low temperature (77 K) fluorescence emission spectrum having three major peaks (at 685, 695, and 730 nanometers when excited at 440 nanometers) which were qualitatively similar to those in the spectrum obtained from mesophyll tissue.These data suggest that S. cernua guard cell chloroplast photosystems I and II contribute to light-dependent stomatal activity only at high light intensities.

Journal Article↗
PubMed2002

Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals.

Caged compounds in combination with protein crystallography represent a valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed close to room temperature. Synchronous reaction initiation has only been achieved with enzymes of relatively slow turnover (<0.1 s(-1)) and caged compounds of high quantum yield. Here X-ray crystallography and microspectrophotometry were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds can be photolyzed by UV light at cryotemperatures. NPE-caged ATP in flash-cooled crystals of Mycobacterium tuberculosis thymidylate kinase was photolyzed successfully at 100-150 K as assessed by the structural observation of ATP-dependent enzymatic conversion of TMP to TDP after temporarily warming the crystals to room temperature. A new method is proposed in which cryo-photolysis combined with temperature-controlled protein crystallography can be used to trap reaction intermediates even in some of the fastest enzymes and/or when only compounds of low quantum yield are available. Raising the temperature after cryophotolysis may allow a transition barrier to be passed and an intermediate to accumulate in the crystal. A comparable method has only been used so far with proteins displaying endogenous photosensitivity. The approach described here opens the way to studying the reaction mechanisms of a much larger number of crystalline enzymes. Furthermore, it is shown that X-ray-induced radiolysis of caged compounds occurs if high-intensity synchrotron beamlines are used. This caveat should be taken into account when deriving data-collection protocols. It could also be used potentially as a way to trigger reactions.

Adenosine Triphosphate↗
PubMed2000

Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals.

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of alpha and beta subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of alpha and beta hemes separately. Previous studies have characterized the oxygen binding by alpha(Ni2+)2beta(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of alpha(Fe2+)2beta(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 +/- 3 and 87 +/- 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 +/- 0.06 and 0.90 +/- 0.03. Analysis of the binding curves, taking into account the different projections of the alpha hemes along the optical directions, indicates that the oxygen affinity of alpha1 hemes is 1.3-fold lower than alpha2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the beta subunits of alpha(Ni2+)2beta(Fe2+)2 crystals. The average oxygen affinity of the alpha subunits (p50 = 91 Torr) is about 1.2-fold higher than the beta subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.

Crystallization↗
PubMed1979

Some aspects of the biology of multinucleate and giant mononucleate erythroblasts in a patient with CDA, type III.

Marrow cells of a patient with CDA, type III, were (1) incubated with 3H-thymidine for 0.5 h and studied using a combination of Feulgen microspectrophotometry and light microscope autoradiography and (2) incubated with 3H-thymidine, 3H-uridine or 3H-leucine for 1 h and studied using the technique of electron microscope autoradiography. The data revealed multiple abnormalities in the proliferation of erythroblasts but no abnormality in the distribution of neutrophil promyelocytes and myelocytes in the different stages of interphase. Some mononucleate erythroblasts had DNA contents of 4-20c and the multinucleate erythroblasts had total DNA contents of 2-40c. In about 40% of the multinucleate erythroblasts, only some of the nuclei within the same cell incorporated 3H-thymidine and the electron microscope autoradiographic studies revealed that the nuclei which failed to synthesize DNA virtually always showed abnormalities in the electron-density of the heterochromatin or euchromatin or a Swiss-cheese appearance of the heterochromatin. Furthermore, in some multinucleate cells but not in others, the ultrastructurally abnormal nuclei showed a marked depression of RNA synthesis when compared with the normal-looking nuclei within the same cell. An occasional binucleate, multinucleate and giant mononucleate erythroblast showed ultrastructural changes suggestive of advanced degeneration and such cells, which showed a marked depression of RNA and protein synthesis, appeared to be phagocytosed by macrophages.

Anemia↗
PubMed1981

Entry of malaria parasites into resealed ghosts of human and simian erythrocytes.

Whereas "white' ghosts, conventionally prepared by hypotonic lysis and washing are resistant to invasion by malaria parasites, cells lysed at high haematocrit and resealed without separating the membranes from the cytosol are invaded with high efficiency (not less than one-third the frequency of intact cells). Similar results were obtained with human and monkey cells, exposed respectively to Plasmodium falciparum and P. knowlesi. Microspectrophotometry of individual cells was used to ascertain the the susceptibility to invasion of lysed, resealed cells was not related to its haemoglobin content.

Animals↗
PubMed1981

Morphology and kinetics of erythropoiesis in haemoglobin H disease.

Some aspects of erythroblast function have been investigated in two Thai patients with haemoglobin H disease. Electron microscope and high resolution autoradiographic studies revealed that (1) some erythroblasts and marrow reticulocytes contained branching intracytoplasmic inclusions and (2) the presence of such inclusions did not necessarily prevent the entry of cells either from the G1 phase into the S phase or from the G2 phase into mitosis. The distribution of early polychromatic erythroblasts in the different stages of the cell cycle was determined by the technique of combined Feulgen microspectrophotometry and 3H-thymidine autoradiography and found to be essentially normal. It is concluded that although the presence of an excess of alpha-chains in the beta-thalassaemia syndromes has been previously shown to be associated with a gross impairment of erythroblast proliferation, the presence of an excess beta-chains in HbH disease is not.

Adult↗
PubMed1984

Ultrastructure and cell cycle distribution of erythropoietic cells in heterozygotes and homozygotes for haemoglobin E.

Marrow aspirates from heterozygotes and homozygotes for haemoglobin E (HbE) have been studied by electron microscopy and by the technique of combined Feulgen microspectrophotometry and 3H-thymidine autoradiography. The erythropoietic cells of heterozygotes did not contain any precipitated globin chains and the proliferating erythroblasts of such individuals showed no abnormality in their distribution in the different stages of interphase. By contrast, 0-1.5% of late erythroblast profiles and 3.1-12.8% of marrow reticulocyte profiles of homozygotes contained intracellular inclusions resembling precipitated alpha-chains. Although precipitated globin chains were not seen in the early polychromatic erythroblasts of homozygotes, the number of these cells in the G2 phase relative to that in the S phase was increased. These data indicate that there is probably little or no imbalance of globin chain synthesis in heterozygotes, a substantial degree of imbalance in homozygotes, and a disturbance of erythroblast proliferation in homozygotes which cannot be attributed to the deleterious effects of detectable intracellular alpha-chain precipitates. The electron microscope and cell cycle distribution data in the homozygotes for HbE were similar to those in two heterozygotes for beta thalassaemia.

Bone Marrow↗
PubMed1991

Congenital dyserythropoietic anaemia with novel intra-erythroblastic and intra-erythrocytic inclusions.

A hitherto undescribed form of congenital dyserythropoietic anaemia is reported. The patient was severely anaemic and hydropic at birth and is now 8 years old. She has a moderate normochromic normocytic anaemia. HbF level of 50%, reticulocyte count of 5-12% and hyperbilirubinaemia. Bone marrow smears showed intense normoblastic erythroid hyperplasia with morphological evidence of dyserythropoiesis; the most common dysplastic features were basophilic stippling of polychromatic erythroblasts and erythrocytes and marked abnormalities of nuclear shape in polychromatic erythroblasts. Electron microscope studies showed that some polychromatic erythroblasts and several erythrocytes contained inclusions which were rounded, elongated or irregular in outline or were doughnut-shaped. These inclusions consisted of compact masses of tubules and saccules which may represent smooth endoplastic reticulum together with Golgi cisternae. The ultrastructural studies also revealed peculiar membrane-bound cylindrical structures in a rare late erythroblast, and phagocytosed erythroblasts within some macrophages. The technique of combined Feulgen microspectrophotometry and 3H-thymidine autoradiography demonstrated a pile-up of early polychromatic erythroblasts in the G1 and G2 phases of the cell cycle, indicating a prolongation of, or an arrest at, these phases. Furthermore, nearly a quarter of all erythroblasts failed to incorporate 3H-leucine into protein. Thus the anaemia appeared to be due to a combination of disordered erythroblast function, increased ineffectiveness of erythropoiesis and peripheral haemolysis. The primary defect may be an excessive synthesis or impaired degradation of intracytoplasmic membranes.

Anemia, Dyserythropoietic, Congenital↗
PubMed1991

Immunomagnetic bead isolation of megakaryocytes from guinea-pig bone marrow: effect of recombinant interleukin-6 on size, ploidy and cytoplasmic fragmentation.

Guinea-pig bone marrow megakaryocytes were isolated using an antibody to platelet glycoprotein Ib and a second antibody conjugated to magnetic beads. The procedure yielded an average of 644,800 megakaryocytes from two guinea-pigs with an average viability of 83%. All of the platelet glycoprotein Ib positive cells also expressed the platelet glycoprotein IIb-IIIa complex. The size and ploidy of megakaryocytes isolated by this technique were analysed in the presence of 10 ng/ml of interleukin-6 (IL-6). Without IL-6 megakaryocyte size increased significantly after 24 h, but an even larger increase in size occurred in the presence of IL-6. The modal ploidy class was 16N with an average of 19% 2N, 2.6% 4N, 16.4% 8N, 50.8% 16N and 11.1% 32N cells as determined by flow cytometry. Measurements made by microspectrophotometry were in close agreement. After 24 h incubation there was a significant rise in the percentage of 2N and 32N cells. The ploidy distribution after 24 h with IL-6 was the same as the control. Megakaryocytes cultured in the absence of serum on collagen gels did not form pseudopods and fragment, as occurs with serum (Leven et al, 1987). Addition of IL-6 to the serum-free cultures caused megakaryocytes to form extensive proplatelet extensions. We conclude that large numbers of pure guinea-pig bone marrow megakaryocytes can be isolated by immunomagnetic bead selection, including low ploidy immature megakaryocytes. Spontaneous maturation occurred as evidenced by the increase in megakaryocyte size and ploidy. IL-6 altered megakaryocyte size and morphology but not ploidy, indicating that these different characteristics of megakaryocytes may be regulated separately.

Animals↗
PubMed1988

Ultrastructure and cell cycle distribution of bone marrow cells in protein-energy malnutrition.

Bone marrow aspirates from four children with kwashiorkor and three with marasmus were studied using the techniques of electron microscopy and combined Feulgen microspectrophotometry and 3H-thymidine autoradiography. The majority of the erythroblasts were ultrastructurally normal, the distribution of the early polychromatic erythroblasts between the various stages of the cell cycle was normal or almost normal, and the macrophages did not contain ingested erythroblasts. Since erythropoietin production has been shown to be normal in protein-energy malnutrition, these findings suggest that at least in some cases of PEM the impairment of erythropoiesis results primarily from an abnormality in the erythroid progenitor cell pool rather than from dyserythropoiesis and ineffective erythropoiesis. In one afebrile and apparently uninfected patient with marasmus, a substantial proportion of the neutrophil granulocytes and their more mature precursors contained electron-dense, myelin-containing intracytoplasmic structures which were presumed to be abnormal primary granules. In four of the patients, the 3H-thymidine labelling index of the neutrophil promyelocyte-myelocyte pool was increased. In addition, in all of the cases, neutrophils at various stages of degradation were readily found within the cytoplasm of some of the macrophages. Thus, whereas the techniques employed did not reveal a major disturbance in the morphologically recognizable precursor cells of the erythroid series in PEM, they demonstrated some abnormalities in such cells of the neutrophil series.

Autoradiography↗
PubMed1991

Chromatin alterations induced by freeze-thawing influence the fertilizing ability of human sperm.

A previous cytochemical study revealed that chromatin alterations could be induced in frozen-thawed human sperm. In order to evaluate the biological consequences of such chromatin alterations, we evaluated the relationship between these chromatin alterations and the drastic decrease or loss of sperm fertilizing ability after freeze-thawing. Using acridine orange staining and Feulgen-DNA quantitative microspectrophotometry, the nuclear variables of sperm were compared using samples which had either recovered, or lost their fertilizing ability after freezing-thawing during artificial inseminations, despite good post-thaw motility. There was a clear decrease in Feulgen-DNA content and nuclear surface values in sperm which exhibited a loss of fertilizing ability. Thus freeze-thawing may alter the DNA/nuclear protein relationships and impair the fertilizing ability of human sperm.

Acridine Orange↗
PubMed1976

DNA synthesis by hybrid echinoid embryos produced from parent species of low and high temperature tolerance.

Synthesis of DNA, as measured by quantitative Feulgen microspectrophotometry, was studied in reciprocal echinoid hybrids and their homospermic controls. Both parent species develop at the same rate until hatching, at 20 degrees C. One parent (Lytechinus) will develop normally at 25 degrees C, a temperature lethal for the other parent employed in the studies (Strongylocentrotus). Strongylocentrotus will develop normally at 10 degrees C, a temperature at which Lytechinus fertilised eggs live, but fail to cleave. It was found that sperm of Lytechinus in eggs of Strongylocentrotus (SL hybrid) show a slowing of the S phase of the cell cycle at 10 degrees C, and that sperm of Strongylocentrotus in eggs of Lytechinus (LS hybrid) show a slowing of the S phase at 25 degrees C. These slowing effects are not noted during cleavage stages. The results are discussed in terms of proteins produced during the time of active transcription of the hybrid genome which may be rate-limiting in the synthesis of DNA in the hybrid embryos under these temperature extremes.

Animals↗
PubMed1988

Hyaluronate accumulation and nerve-dependent growth during regeneration of larval Ambystoma limbs.

Hyaluronate-mediated expansion of the extracellular matrix has been suggested as an important element of growth and morphogenesis in several developing systems. In vitro, various growth factors have been shown to stimulate hyaluronate synthesis as well as cell proliferation. A similar link between proliferation and hyaluronate production during in vivo growth is difficult to demonstrate, because in most systems the source of growth-promoting factors is either not known or not amenable to experimental manipulation. During amphibian limb regeneration, cell proliferation depends upon paracrine release of factors from axons in the limb stump, and the nerve supply can be eliminated or augmented experimentally for study of growth in this system. Denervated and amputated limbs of larval salamanders do not begin to regenerate until distal areas of the limb stumps are reinnervated. We have used such limbs to examine the effect exerted by the reappearance of nerves on the amount of hyaluronate in the tissue undergoing the growth response. Hyaluronate was demonstrated by the metachromatic dye Ethyl Stains-all, which stains hyaluronate blue while sulfated glycosaminoglycans (GAGs) and proteins in the extracellular matrix stain various shades of violet, and by microspectrophotometry of alcian-blue-stained GAGs in serial sections pretreated with buffer or with Streptomyces hyaluronidase (SH) to remove hyaluronate specifically. Both methods showed little hyaluronate in the distal region of limb stumps prior to reinnervation, while reinnervated stumps had amounts of hyaluronate similar to those of control blastemas. Autoradiography of 3H-glucosamine-labeled limbs indicated that hyaluronate in the blastemas of reinnervated limb stumps included material newly synthesized by cells throughout the growing tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Ambystoma↗
PubMed1979

Catalytic activity of aspartate aminotransferase in the crystal. Equilibrium and kinetic analysis.

Natural substrates and analogs rapidly diffuse through crystals of pig heart mitochondrial aspartate aminotransferase and react at the active sites causing spectral changes that can be measured by single-crystal microspectrophotometry. Dissociation constants for natural substrates and rate constants of transamination for slowly reacting substrates have been determined. A comparison between the data obtained in the crystal and in solution shows that the crystalline enzyme is catalytically competent and that events occurring in the crystal essentially parallel those occurring in solution, even though minor differences have been detected.

Animals↗
PubMed1994

Effect of increased myocardial cyclic GMP induced by cyclic GMP-phosphodiesterase inhibition on oxygen consumption and supply of rabbit hearts.

1. We tested the hypothesis that increasing myocardial cyclic GMP levels would reduce myocardial O2 consumption and areas of low O2 supply/consumption balance, using zaprinast, a selective cyclic GMP-phosphodiesterase inhibitor. 2. The study was conducted in three groups (vehicle, 10(-3) and 3 x 10(-3) mol/L zaprinast) of anaesthetized open-chest New Zealand white rabbits (n = 24). Coronary blood flow (radioactive microspheres), arterial and venous O2 saturation (microspectrophotometry), O2 consumption, cyclic GMP content (competitive binding) and cyclic GMP-phosphodiesterase activity (conversion of 3H-cyclic GMP to 3H-GMP) were determined. 3. Agents were applied to a patch on the myocardial surface and did not cause significant haemodynamic changes, except for bradycardia in the vehicle and low dose group. 4. The total myocardial cyclic GMP-phosphodiesterase activity was 148 +/- 14 while the zaprinast (10 mumol/L) inhibitable activity averaged 63 +/- 8 pmol/mg protein per min. Cyclic GMP content was increased with increasing doses of zaprinast (vehicle, 4.308 +/- 0.349 pmol/g; low dose zaprinast, 4.803 +/- 0.279 and high dose zaprinast, 7.938 +/- 1.304 pmol/g). 5. Coronary blood flow was not different after treatment (198 +/- 11, 209 +/- 10 and 153 +/- 9 mL/min per 100 g for the vehicle, low and high dose zaprinast, respectively). 6. Under control conditions, 48% of the small veins had O2 saturations below 50%. With zaprinast, this value was reduced to 19% for the low and 24% for the high dose. 7. Average venous O2 saturation increased with zaprinast (49 +/- 2%, 61 +/- 3% and 59 +/- 1%).(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1977

Euglena: the photoreceptor system for phototaxis.

The photoreceptor structures (eyespot-paraflagellar body-flagellum) for Euglena phototaxis were investigated by electron microscopy. The paraflagellar body--the photoreceptor--is a highly ordered crystalline lamellar structure. Optical diffraction of the electron micrographs and resulting filtered images of the paraflagellar body suggest that it is formed of rods in a helical arrangement. The action spectra for phototaxis, the in situ spectrum by microspectrophotometry of the paraflagellar body, and flavin analysis of the organism indicate that the photoreceptor molecule is a flavoprotein. The phototaxis action spectrum is similar to the spectrum for O2 evolution and implies that similar molecules participate in the photo processes. As a result, a photochemical scheme is suggested in which a photo-excited flavin and a cytochrome participate in the photoprocess. The photochemistry and photoreceptor structures for Euglena phototaxis are likened to a photoneuro sensory cell.

Animals↗
PubMedDate not supplied

Genotoxic effects of linear alkyl benzene sulfonate, sodium pentachlorophenate and dichromate on Tetrahymena pyriformis.

DNA in macro- and micronuclei of Tetrahymena pyriformis treated with linear alkyl benzene sulfonate (LAS) and sodium pentachlorophenate (PCP-Na) were determined by microspectrophotometry. The effects on rate of formation of macronuclear DNA extrusion bodies were also studied. We found DNA content of micronuclei in 0.14 ppm LAS and 0.9 ppb PCP-Na was lower than in that of the control, and LAS was able to increase the formation rate of macronuclear DNA extrusion bodies (the formation rate was 54% in 11.3 ppm LAS and 25.6% in 16.7 ppm dichromate). We concluded that 0.14 ppm LAS (below the maximum acceptable toxicant concentration) was genotoxic, whereas 0.014 ppm LAS was not. Dichromate 0.05 ppm and 0.9 ppb PCP-Na, equal to and below the maximum acceptable toxicant concentration, respectively, were potentially genotoxic.

Alkanesulfonates↗