Non-photochemical quenching in the cells of the carotenogenic chlorophyte Haematococcus lacustris under favorable conditions and under stress.

The microalga Haematococcus lacustris (formerly H. pluvialis) is the richest source of the valuable pigment astaxanthin, accumulated in red aplanospores (haematocysts). In this work, we report on the photoprotective mechanisms in H. lacustris, conveying this microalga its ability to cope with a wide range of adverse conditions, with special emphasis put on non-photochemical quenching (NPQ) of the excited chlorophyll states. We studied the changes in the primary photochemistry of the photosystems (PS) as a function of irradiance and the physiological state. We leveraged the transcriptomic data to gain a deeper insight into possible NPQ mechanisms in this microalga. Peculiar to H. lacustris is a bi-phasic pattern of changes in photoprotection during haematocyst formation. The first phase coincides with a transient rise of photosynthetic activity. Based on transcriptomic data, high NPQ level in the first phase is maintained predominantly by the expression of PsbS and LhcsR proteins. Then, (in mature haematocysts), stress tolerance is achieved by optical shielding by astaxanthin and dramatic reduction of photosynthetic apparatus. In contrast to many microalgae, shielding plays an important role in H. lacistris haematocysts, whereas regulated NPQ is suppressed. Astaxanthin is decoupled from the PS, hence the light energy is not transferred to reaction centers and dissipates as heat. It allows to retain a higher photochemical yield in haematocysts comparing to vegetative cells. The ability of H. lacustris to substitute the "classical" active photoprotective mechanisms such as NPQ with optic shielding and general metabolism quiescence makes this organism a useful model to reveal photoprotection mechanisms.

Suitability of Anabaena PCC7120 expressing mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis for biotechnological application.

We present evidence that Anabaena PCC7120 (A.7120) strains expressing mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis (Bti) have a strong potential for biotechnological application. Characterization of two 4-year-old recombinant A.7120 clones constructed previously in our laboratory [clone 7 and clone 11, each carrying three Bti genes (cry4Aa, cry11Aa, and p20)] revealed three facts. First, the Bti genes were stable in A.7120 even in the absence of antibiotic selection when the genes were integrated in the chromosome (in clone 11); and the genes were also stable as plasmid-borne constructs (in clone 7), provided the cultures were maintained under continued selection. Second, clone 7 (kept under selection) and clone 11 (either kept or not kept under selection) continued to be mosquitocidal through 4 years of culture. Third, growth of the recombinant clones was comparable to the wild type under optimal growth conditions, indicating that growth was not compromised by the expression of toxin genes. These results clear the way for the development of mass production techniques for A.7120 strains expressing Bti toxin genes.

Presence of a nonhydrolyzable biopolymer in the cell wall of vegetative cells and astaxanthin-rich cysts of Haematococcus pluvialis (Chlorophyceae).

The green alga Haematococcus pluvialis accumulates massive amounts of the red pigment astaxanthin in response to stimuli inducing it to form cysts. During the encystment process the cell wall undergoes a clear hardening and thickening. In this work, a cell wall fraction withstanding successive acid and basic hydrolysis was isolated and proves to be algaenan by Fourier transform infrared spectroscopy. This compound is equally abundant in nonmotile vegetative cells and astaxanthin-rich cysts. This finding indicates that the synthesis of algaenan does not require the activation of the machinery for the massive production of secondary carotenoids. We conclude that algaenan cannot cause the changes occurring in the cell wall during the encystment process without the involvement of other cell wall components.

The effect of B-mode ultrasonic image standardisation on the echodensity of symptomatic and asymptomatic carotid bifurcation plaques.

BACKGROUND: To develop a method that allows B-mode ultrasonic images of carotid plaques to be standardised so that measurements of plaque echodensity become comparable and clinically useful. METHODS: Design. Cross sectional study. Setting. Teaching hospital, England. PATIENTS: a random consecutive series of 23 patients in part I, 19 in part II and 77 in part III. Measures. Part I: twenty-three images of carotid bifurcation plaques from 2 duplex scanners were digitised. Images were standardised by 4 observers so that the grey scale median (GSM) would be 0-5 for blood and 185-195 for adventitia. Part II: the effect of three different recording media: video, magneto-optical disk (MOD) and thermal paper on the echodensity of 19 plaques' images was determined. Part III: the echodensity of 91 carotid bifurcation plaques with greater than 50% stenosis was correlated to the presence or absence of ipsilateral hemispherical symptoms. RESULTS: Part I: the coefficient of variation (CV) among 4 observers was 0.7%, 0% and 4.7% after image standardisation for the adventitia, blood and plaques respectively. Part II: a near perfect agreement was obtained between the GSM of plaques from images on video and MOD (r = 0.97) after standardisation. Part III: after standardisation, the GSM of symptomatic plaques was lower (21 +/- 14.8) than in asymptomatic plaques (38 +/- 26) p = 0.002. Plaque echolucency was more likely to be associated with symptoms (relative risk 4.1 90% CI 1.8-9.4). CONCLUSIONS: Images from different scanners by different ultrasonographers and through different peripherals can be standardised so that measurements of plaque echodensity may become comparable. The method is recommended for use in future natural history studies on carotid plaques where stroke is the end-point.

Does astaxanthin protect Haematococcus against light damage?

The photoprotective function of the ketocarotenoid astaxanthin in Haematococcus was questioned. When exposed to high irradiance and/or nutritional stress, green Haematococcus cells turned red due to accumulation of an immense quantity of the red pigment astaxanthin. Our results demonstrate that: 1) The addition of diphenylamine, an inhibitor of astaxanthin biosynthesis, causes cell death under high light intensity; 2) Red cells are susceptible to high light stress to the same extent or even higher then green ones upon exposure to a very high light intensity (4000 mumol photon m(-2)s(-1)); 3) Addition of 1O2 generators (methylene blue, rose bengal) under noninductive conditions (low light of 100 mumol photon m(-2)s(-1) induced astaxanthin accumulation. This can be reversed by an exogenous 1O2 quencher (histidine); 4) Histidine can prevent the accumulation of astaxanthin induced by phosphate starvation. We suggest that: 1) Astaxanthin is the result of the photoprotection process rather than the protective; 2) 1O2 is involved indirectly in astaxanthin accumulation process.

[Granulomatous process of the optic papilla as an initial sign of systemic sarcoidosis]. / Granulomatöser Papillenprozess als erstes Zeichen einer systemischen Sarkoidose.

Sarcoidosis is a systemic granulomatosis disease of unknown cause that may involve many ocular structures in about one third of cases. The findings of a posterior uveitis include vitritis, retinal periphlebitis, chorioretinal infiltrates, serpiginouschoroiditis, optic nerve atrophy and edema or granulomatous infiltration of the papilla. The central nervous system is clinically affected in 5 to 15% of cases. Despite several dozen published case reports of sarcoidoses of the optic nerve, direct infiltration of this structure is considered rare. We describe herein the case of 25-year-old white man whose only symptom of systemic sarcoidosis was optic nerve granuloma.

Intravascular ultrasound imaging after excimer laser angioplasty.

To help elucidate the mechanism of excimer laser coronary angioplasty (ELCA), intravascular ultrasound (IVUS) imaging was performed in 19 of 29 patients who were treated with ELCA. The results were compared with a non-randomized control group of 18 patients who had IVUS studies both before and after PTCA alone. After ELCA alone, lumen diameter (1.9 x 1.7 mm) and lumen cross-sectional area (CSA) (2.9 mm2) by IVUS were not significantly different from baseline values in the patients before PTCA alone (2.1 x 1.8 mm, 3.2 mm2). After balloon dilatation in the laser treated group, lumen diameter (2.5 x 2.1 mm) and lumen CSA (4.9 mm2) were significantly greater than those post ELCA alone. However, there was no difference in lumen CSA or atheroma CSA in the group treated with excimer laser plus balloon dilatation vs. these measurements in the group treated with PTCA alone. ELCA does not ablate a large amount of atheroma (9% reduction) but creates a pathway to permit easier passage of a PTCA balloon. These quantitative and morphologic results may help explain why the restenosis rate with ELCA is similar to PTCA alone.

Comparison of the natural history of irregular and smooth coronary lesions: insights into the pathogenesis, progression, and prognosis of coronary atherosclerosis.

The coronary arteriograms of 255 patients who had two to four arteriograms within 2.6 +/- 1.7 years were reviewed. Two hundred three patients had lesions on at least one arteriogram; among the 167 patients without coronary surgery, there were 48 complex irregular lesions (suggesting a ruptured plaque and/or thrombosis) and 141 smooth lesions with follow-up, and 73 irregular and 164 smooth lesions with preceding arteriograms available. Severe irregular lesions (> or = 90% diameter occlusion) progressed to total occlusion (46%) more often than did severe smooth lesions (11.5%) (p < 0.01). Less severe lesions usually did not progress, with no difference in incidence of progression between irregular and smooth lesions (27.8% vs 23.9%). Irregular lesions > or = 80% usually occurred as a result of progression in less severe smooth lesion or occurred in areas that were minimally diseased or appeared normal, whereas smooth lesions > or = 80% had usually not changed since the previous arteriogram. Irregular lesions very rarely became smooth. A study of lesions in 36 patients with surgery was confirmatory. We conclude that plaque rupture is a common mechanism for progression of coronary disease but is not a common pathway for the growth of smooth lesions; irregular lesions remain irregular for years. There is no relationship between the severity of smooth plaques and their likelihood to rupture. Progression of coronary disease can occur by either of two modes: (1) gradual growth of a smooth-walled plaque or (2) plaque rupture with marked progression to a severe irregular lesion. Because most smooth and most irregular lesions remain stable for years, except possibly for > or = 90% irregular lesions, there is no anatomic finding that justifies urgent revascularization. Instability is a clinical diagnosis.

Characterization and photoaffinity labeling of the ATP binding site of the ryanodine receptor from skeletal muscle.

The photoaffinity analog of ATP, 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (Bz2ATP) was used to covalently label and to identify the ATP binding site of the skeletal muscle ryanodine receptor. Like ATP, Bz2ATP stimulates up to fivefold the binding of ryanodine to its receptor. Photoactivation by ultraviolet light of the benzophenone group in the [alpha-32P]Bz2ATP results in covalent binding of [alpha-32P]Bz2ATP to the 450-kDa polypeptide, the ryanodine receptor's subunit. An apparent molar stiochiometry of Bz2ATP to the tetrameric ryanodine receptor complex of 1.146 +/- 0.087 (n = 2) was estimated. The covalent binding of [alpha-32P]Bz2ATP was inhibited by ATP and analogous compounds in the order: ATP = AdoPP[CH2]P = ADP = Ado = cAMP > AMP > ITP = GTP. Similar specificity was obtained for the stimulation of ryanodine binding by these nucleotides. ATP increased the ryanodine binding affinity by about sixfold. The polycationic dye ruthenium red, known as an inhibitor of Ca2+ release and ryanodine binding, inhibited the labeling of the ryanodine receptor by [alpha-32P]Bz2ATP. Tryptic digestion of the ryanodine receptor revealed a [alpha-32P]Bz2ATP-labeled 76-kDa tryptic fragment. Digestion of either the [alpha-32P]Bz2ATP-labeled 450-kDa or the 76-kDa polypeptides with S. aureus resulted in the appearance of four labeled fragments of 39, 33, 27 and 13 kDa, where the 39-kDa fragment is the precursor of the 27-kDa and 13-kDa fragments. The results suggest that the regulation of Ca2+ release by ATP involves an ATP binding site(s) located on the 27-kDa and 13-kDa fragments of the ryanodine receptor protein.

The interaction of spermine with the ryanodine receptor from skeletal muscle.

The effect of polyamines on ryanodine binding activity of junctional sarcoplasmic reticulum membranes is described. Spermine stimulated the binding of ryanodine to its receptor up to 5-fold, with half-maximal stimulation obtained with 3.5 mM. Spermidine and putrescine also stimulated ryanodine binding, but they were about 12-fold less potent. The degree of stimulation is dependent on the NaCl concentration present in the assay medium. Spermine has no effect on the Ca(2+)-dependency of ryanodine binding but it increases the ryanodine binding affinity (Kd) by about 5.6-fold. Both the rate of ryanodine association with, and dissociation from, its binding site were affected by spermine. Spermine also stimulates the photoaffinity labelling by 3-O-(4-benzoyl)benzoyl[alpha-32P]ATP ([alpha-32P]BzATP) of the ryanodine receptor, increasing the BzATP binding affinity. We suggest that the stimulatory effect of spermine on ryanodine binding is due to its specific interaction with the ryanodine receptor. This spermine interaction enabled us to develop a new, one-step, fast and with high yield method for the purification of ryanodine receptor (Shoshan-Barmatz, V. and Zarka, A. (1992) Biochem. J. 284, in press).

A simple, fast, one-step method for the purification of the skeletal-muscle ryanodine receptor.

In this paper we describe a simple, fast, one-step method for the purification of the skeletal-muscle ryanodine receptor. The ryanodine receptor from CHAPS-solubilized junctional sarcoplasmic-reticulum membranes was adsorbed to a spermine-agarose column and eluted by 2 mM-spermine. The purified receptor, consisting predominantly of a 450 kDa polypeptide on SDS/PAGE, binds [3H]ryanodine with a specific activity of approximately 300 pmol/mg of protein and with a high affinity (KD = 4.7 +/- 2 nM). The purified receptor appears to retain the pharmacological properties of the receptor in the original membranes. The purification resulted in over 80% recovery of the initial ryanodine-binding sites and about 30-96-fold purification. This simple and fast method is highly reproducible and suitable for purification of small as well as large quantities of ryanodine receptor.

Morphological effects of coronary balloon angioplasty in vivo assessed by intravascular ultrasound imaging.

BACKGROUND: Histological examination of the effects of balloon angioplasty have been described from in vitro experiments and a limited number of pathologic specimens. Intravascular ultrasound imaging permits real time cross-sectional observation of the effect of balloon dilation on the atherosclerotic plaque in vivo. METHODS AND RESULTS: The morphological effects of coronary angioplasty were visualized at 66 lesions in 47 patients immediately after balloon dilatation with an intravascular ultrasound imaging catheter. Cross-sectional images were obtained at 30 frames per second as the catheter passed along the length of the artery. Quantitative and qualitative assessments of the dilated atherosclerotic plaque were made from the angiograms and the ultrasound images. Six morphological patterns after angioplasty were appreciated by ultrasound imaging. Type A consists of a linear, partial tear of the plaque from the lumen toward the media (seven lesions); Type B is defined by a split in the plaque that extends to the media (12 lesions); Type C demonstrates a dissection behind the plaque that subtends an arc of up to 180 degrees around the circumference (18 lesions); Type D was a more extensive dissection that encompasses an arc of more than 180 degrees (four lesions); and Type E may be present in either concentric (Type E1, 14 lesions) or eccentric (Type E2, 11 lesions) plaque and is defined as an ultrasound study without any evidence of a fracture or a dissection in the plaque. There was a large amount of residual atheroma in each type of morphology (7.8 +/- 2.9 mm2, 61.6 +/- 15.4% of cross-sectional area); there was no difference, however, in lumen or atheroma cross-sectional area among these six patterns. There was a good correlation between ultrasound and angiography for the recognition of a dissection. Calcification was seen in only 14% of lesions on angiography, whereas most lesions (83%) revealed calcification on ultrasound imaging. As determined by intravascular ultrasound, calcified plaque was more likely to fracture in response to balloon dilatation than noncalcified plaque (p less than 0.01). Thirteen of 66 lesions (20%) developed clinical and angiographic restenosis. Restenosis was more likely to occur when the original dilatation left a concentric plaque without a fracture or dissection (Type E1, 50% incidence) compared with a mean restenosis rate of 12% in the remaining morphological patterns (p = 0.053). CONCLUSIONS: Intravascular ultrasound provides a more complete quantitative and qualitative description of plaque geometry and composition than angiography after balloon angioplasty. In addition, intravascular ultrasound identified a subset of atherosclerotic plaque that has a higher incidence of restenosis. This information could be used prospectively to consider other therapeutic options in this subset. Intravascular ultrasound provides a method to describe the effects of angioplasty that will be useful in comparing future coronary intervention studies.

Thickness-shear mode shapes and mass-frequency influence surface of a circular and electroded AT-cut quartz resonator.

Finite-element solutions for the fundamental thickness shear mode and the second-anharmonic overtone of a circular, 1.87-MHz AT-cut quartz plate with no electrodes are presented and compared with previously obtained results for a rectangular plate of similar properties. The edge flexural mode in circular plates, a vibration mode not seen in the rectangular plate is also presented. A 5-MHz circular and electroded AT-cut quartz plate is studied. A portion of the frequency spectrum is constructed in the neighborhood of the fundamental thickness-shear mode. A convergence study is also presented for the electroded 5-MHz plate. A new two-dimensional (2-D) technique for visualizing the vibration mode solutions is presented. This method departs substantially from the three-dimensional (3-D) ;wire-frame' plots presented in the previous analysis. The 2-D images can be manipulated to produce nodal line diagrams and can be color coded to illustrate mode shapes and energy trapping phenomenon. A contour plot of the mass-frequency influence surface for the plated 5-MHz resonator is presented. The mass-frequency influence surface is defined as a surface giving the frequency change due to a small localized mass applied to the resonator surface.

The origin and fate of complex coronary lesions.

Complex irregular coronary artery stenoses, representing plaque rupture/thrombosis, are associated with the acute coronary syndromes. However, the natural history (origin and fate) of these lesions is not known. To examine this issue we studied 255 patients who had had two to four arteriograms within a mean interval of 2.6 +/- 1.7 years. Of 53 irregular lesions that had progressed on a later arteriogram, 35 (66%) originated from areas that were smooth and less than 50% in stenosis diameter. Of 44 irregular lesions on an earlier study, 10 (23%) became totally occluded, five (11%) progressed in severity (all remained irregular), 25 (57%) showed no change in severity (all remained irregular), and four (9%) regressed (two became smooth). Nine of the 10 lesions progressing to occlusion were greater than or equal to 95% stenosed on the earlier study. Only 2 of 44 lesions (5%) showed smoothing. These findings are in agreement with the concept that irregular lesions represent ruptured atherosclerotic plaques and demonstrate that they usually originate from mildly occlusive smooth plaques. Markedly narrowed irregular lesions (greater than or equal to 95% stenosis) frequently progress to occlusion. Irregular lesions less than 90% narrowed commonly remain angiographically stable, and irregular over several years. They were found rarely to evolve into smooth-walled plaques.

Trypsin destruction of the high affinity ryanodine binding sites of the junctional sarcoplasmic reticulum.

Tryptic digestion of the junctional sarcoplasmic reticulum membranes in sucrose but not NaCl buffer leads to complete loss of ryanodine binding capacity. The presence of MgCl2 in the sucrose buffer prevents the loss of ryanodine binding by the trypsin treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the treated membranes reveal that the 400-kDa protein band disappeared under all the different digestion conditions. However, the presence of 135-kDa tryptic fragment is observed only when ryanodine binding is retained. Quantitative analysis of the gels shows that the loss of ryanodine binding is well correlated with the cleavage of the 135-kDa tryptic fragment. This correlation is obtained when the cleavage was controlled either by the digestion time or by NaCl or MgCl2 concentrations. The same concentrations of MgCl2 and NaCl affect the ryanodine binding activity, the cleavage of the 135-kDa tryptic fragment, and the solubility and stability of the [3H]ryanodine-receptor complex in a detergent-containing medium. Tryptic digestion of the ryanodine receptor/junctional Ca2+ release channel, which leads to complete loss of ryanodine binding capacity, has no effect or slightly stimulates the Ca2+ accumulation activity of these membranes.