Isolation and properties of the luciferase stored in the ovary of the scyphozoan medusa Periphylla periphylla.

Bioluminescence of the medusa Periphylla is based on the oxidation of coelenterazine catalyzed by luciferase. Periphylla has two types of luciferase: the soluble form luciferase L, which causes the exumbrellar bioluminescence display of the medusa, and the insoluble aggregated form, which is stored as particulate material in the ovary, in an amount over 100 times that of luciferase L. The eggs are especially rich in the insoluble luciferase, which drastically decreases upon fertilization. The insoluble form could be solubilized by 2-mercaptoethanol, yielding a mixture of luciferase oligomers with molecular masses in multiples of approximately 20 kDa. Those having the molecular masses of 20 kDa, 40 kDa, and 80 kDa were isolated and designated, respectively, as luciferase A, luciferase B, and luciferase C. The luminescence activities of Periphylla luciferases A, B, and C were 1.2 approximately 4.1 x 10(16) photon/mg. s, significantly higher than any coelenterazine luciferase known, and the quantum yields of coelenterazine catalyzed by these luciferases (about 0.30 at 24 degrees C) are comparable to that catalyzed by Oplophorus luciferase (0.34 at 22 degrees C), which has been considered the most efficient coelenterazine luciferase until now. Luciferase L (32 kDa) could also be split by 2-mercaptoethanol into luciferase A and an accessory protein (approx. 12 kDa), as yet uncharacterized. Luciferases A, B, and C are highly resistant to inactivation: their luminescence activities are only slightly diminished at pH 1 and pH 11 and are enhanced in the presence of 1 approximately 2 M guanidine hydrochloride; but they are less stable to heating than luciferase L, which is practically unaffected by boiling.

Ossification of the posterior longitudinal ligament and ligamentum flavum: imaging features.

Ossifications of the posterior longitudinal ligament and ligamentum flavum are both special subcategories of degenerative diseases responsible for compression of the spinal cord. Ossification of the ligaments is well demonstrated by plain radiography and computed tomography. Magnetic resonance imaging noninvasively provides useful information about the degree and extent of spinal cord compression as well as the character of the ossification. T2-weighted sequences are most effective to evaluate both spinal cord compression due to the ossification and abnormal signal intensity of the spinal cord.

7,8-Dihydropterin-6-carboxylic acid as light emitter of luminous millipede, Luminodesmus sequoiae.

A luminous millipede. Luminodesmus sequoiae, emits light centered at a wavelength of 500 nm. To determine the light emitter of this bioluminescent system, fluorescent compounds were isolated from pulverized cuticles. NMR and MS spectra of these compounds showed them to be pterin derivatives. Furthermore, proton/deuterium (H/D) exchange experiments by ESI-Q-TOF-MS and -MS/MS measurements have proved to be a powerful tool for elucidating these heteroaromatic compounds. Finally, we have concluded that 7,8-dihydropterin-6-carboxylic acid, a new natural product, is the light emitter of Luminodesmus bioluminescence.

Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase(1).

The deep-sea shrimp Oplophorus gracilirostris secretes a luciferase that catalyzes the oxidation of coelenterazine to emit blue light. The luciferase (M(r) approx. 106000) was found to be a complex composed of 35 kDa and 19 kDa proteins, and the cDNAs encoding these two proteins were cloned. The expression of the cDNAs in bacterial and mammalian cells indicated that the 19 kDa protein, not the 35 kDa protein, is capable of catalyzing the luminescent oxidation of coelenterazine. The primary sequence of the 35 kDa protein revealed a typical leucine-rich repeat sequence, whereas the catalytic 19 kDa protein shared no homology with any known luciferases including various imidazopyrazinone luciferases.

The crystal structure of the photoprotein aequorin at 2.3 A resolution.

Aequorin is a calcium-sensitive photoprotein originally obtained from the jellyfish Aequorea aequorea. Because it has a high sensitivity to calcium ions and is biologically harmless, aequorin is widely used as a probe to monitor intracellular levels of free calcium. The aequorin molecule contains four helix-loop-helix 'EF-hand' domains, of which three can bind calcium. The molecule also contains coelenterazine as its chromophoric ligand. When calcium is added, the protein complex decomposes into apoaequorin, coelenteramide and CO2, accompanied by the emission of light. Apoaequorin can be regenerated into active aequorin in the absence of calcium by incubation with coelenterazine, oxygen and a thiol agent. Cloning and expression of the complementary DNA for aequorin were first reported in 1985 (refs 2, 6), and growth of crystals of the recombinant protein has been described; however, techniques have only recently been developed to prepare recombinant aequorin of the highest purity, permitting a full crystallographic study. Here we report the structure of recombinant aequorin determined by X-ray crystallography. Aequorin is found to be a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxide. The structure shows protein components stabilizing the peroxide and suggests a mechanism by which calcium activation may occur.

Light-emitters involved in the luminescence of coelenterazine.

Coelenterazine emits light by chemi-and bioluminescence reactions, decomposing into coelenteramide and CO(2). To ascertain the light emitters involved, the fluorescence of coelenteramide and five analogues were studies in four kinds of solvent. The results showed that coelenteramides can form five kinds of light emitters, ie unionized (lambda(max) 386-423 nm), phenolate anion (lambda(max) 480-490 nm), phenolate anion temporarily formed from the ion-pair state (lambda(max) 465-479 nm), amide anion (lambda(max) 435-458 nm) and pyrazine-N(4) anion (lambda(max) 530-565 nm). The chemiluminescence light emitter of coelenterazine in the presence of alkali (lambda(max) 530-550 nm) was found to be the pyrazine-N(4) anion and not the dianion (ie phenolate anion/amide anion), as previously believed. In chemiluminescence, the normal light emitter is the amide anion, and the pyrazine-N(4) anion emission may occur in the presence of alkali, but light emission from any other emitters has not been observed. In the bioluminescence reaction, the normal light emitter is the amide anion, but no other light emitter was observed except the unionized form found in the Ca-triggered luminescence of semisynthetic aequorins prepared with an e-type coelenterazine instead of coelenterazine.

A first-order liquid-liquid phase transition in phosphorus

First-order structural phase transitions are common in crystalline solids, whereas first-order liquid-liquid phase transitions (that is, transitions between two distinct liquid forms with different density and entropy) are exceedingly rare in pure substances. But recent theoretical and experimental studies have shown evidence for such a transition in several materials, including supercooled water and liquid carbon. Here we report an in situ X-ray diffraction observation of a liquid-liquid transition in phosphorus, involving an abrupt, pressure-induced structural change between two distinct liquid forms. In addition to a known form of liquid phosphorus--a molecular liquid comprising tetrahedral P4 molecules--we have found a polymeric form at pressures above 1 GPa. Changing the pressure results in a reversible transformation from the low-pressure molecular form into the high-pressure polymeric form. The transformation is sharp and rapid, occurring within a few minutes over a pressure range of less than 0.02 GPa. During the transformation, the two forms of liquid coexist. These features are strongly suggestive of a first-order liquid-liquid phase transition.

A new method for crosstalk correction in simultaneous dual-isotope myocardial imaging with Tl-201 and I-123.

We have developed a new method of crosstalk correction in simultaneous dual-isotope imaging with Tl-201 and I-123 by using crosstalk ratios and a blurring filter. Single isotope myocardial studies (10 for Tl-201 and 7 for I-123) were performed with a dual energy window acquisition mode and two low energy general-purpose collimators. Then two planar images acquired with dual energy windows for a Tl-201 line source and an I-123 line source were obtained to measure line spread functions (LSFs) and crosstalk ratios for each image. The line source experiments showed that the LSFs for the Tl-201 imaging window from the single Tl-201 source were very similar to those for the I-123 imaging window from the single Tl-201 source, but the LSFs for the Tl-201 imaging window from the single I-123 source had broad shapes which differed from those for the I-123 imaging window from the single I-123. To obtain accurate I-123 crosstalk images in the Tl-201 imaging window from the I-123 images in the I-123 imaging window, we designed a low-pass blurring filter. In 7 clinical I-123 MIBG studies, I-123 window images processed with this filter became very similar to the Tl-201 window image from the single I-123 source. The method proposed in this study can accurately correct the crosstalk in dual isotope studies with Tl-201 and I-123 and is easily applicable to conventional gamma camera systems with any dual energy window acquisition mode.

The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin.

Recombinant apoaequorin expressed in the periplasmic space of Escherichia coli cells was regenerated into aequorin and extracted from the cells, simultaneously, using a buffer that contained coelenterazine. Due to the mild extraction conditions, the impurities in the extract were minimal. Thus, the purification of extracted aequorin could be accomplished in only two steps, anion-exchange chromatography and hydrophobic interaction chromatography, simply by adsorption and elution in both steps. The purified recombinant aequorin was pure, based on various data, including HPLC analysis and light-emitting activity. The yield of purified aequorin was 25-35 mg from 600 ml of culture, which was over 75% of the total amount of apoaequorin expressed in E. coli cells.

Evaluation of five imidazopyrazinone-type chemiluminescent superoxide probes and their application to the measurement of superoxide anion generated by Listeria monocytogenes.

Superoxide-triggered chemiluminescence of five new imidazopyrazinone derivatives was investigated using the hypoxanthine-xanthine oxidase system as the source of superoxide anion. The results showed that they are highly sensitive and have favorable properties in measuring superoxide anion. With those new probes, the generation of superoxide anion from the bacteria Listeria monocytogenes was examined. The results confirmed the previous report that L. monocytogenes is an unusual organism that extracellularly and continuously generates a high level of superoxide anion in the presence of acetaldehyde. The data indicated that two of the probes, 3,7-dihydro-2-methyl-6-phenylethynylimidazo[1,2-a]pyrazin-3- one (4) and its methoxy derivative (5), are highly sensitive and useful in the measurements of superoxide anion and are clearly superior to 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3-on e (MCLA), which-has been generally considered the most sensitive superoxide probe in the past. When tested at a probe concentration of 3.3 microM, the luminescence response and the signal-background ratio of compound 4 were 1.5 and 2.5 times those of MCLA, respectively, and the signal-background ratio of compound 5 was almost 15 times that of MCLA, though the luminescence response of this compound was slightly lower than that of MCLA. The low probe concentration used enhances the usefulness of probes in the measurements of superoxide in functioning biological systems.

Ossification of posterior longitudinal ligaments: evaluation with MRI.

Ossification of the posterior longitudinal ligament is a special subcategory of degenerative disease responsible for compression of the spinal cord. On MR images, T2-weighted sequences are the most effective to evaluate both spinal cord compression due to the ossification and abnormal signal intensity of the cord. Although ossification of the ligaments is well demonstrated on CT and plain radiographs, MRI noninvasively provides useful information about the degree and extent of spinal cord compression, as well as the character of the ossification.

MR findings of hypoxic damage of the bilateral striate cortices: a case report.

MR imaging of a patient with cortical blindness revealed areas of abnormal signal bilaterally in the lateral putamen and the striate cortices. The coronal T2-weighted image clearly demonstrated the selective involvement of the bilateral striate cortices. The differential diagnosis and the vulnerability of the striate cortex are discussed.

Luciferase of the Scyphozoan Medusa Periphylla periphylla.

Two types of luciferase that catalyze the luminescent oxidation of coelenterazine were isolated from the marginal exumbrella epithelium (lappet) and the ovary of Periphylla periphylla; they were designated luciferase-L and luciferase-O, respectively. Luciferase-L (Mr 32,000), probably derived from highly specialized photocytes, was very resistant to heat, and its activity was little affected by boiling; but it was unstable in solutions of low ionic strength if bovine serum albumin was not included in the solvent. Luciferase-O (Mr 75,000) occurred in the eggs in association with particulate matter, and was solubilized and extracted with a buffer containing 2 M guanidine hydrochloride; the enzyme was highly stable in this strongly denaturing solvent. The intensities of the coelenterazine luminescence catalyzed by both luciferases were maximal at pH 7.8 and in the presence of about 1 M NaCl. The quantum yield of coelenterazine was estimated to be 0.14 with luciferase-L (emission max. at 465 nm) and 0.12 with luciferase-O (emission max. at 470 nm). The luminescence caused by both luciferases was strongly inhibited by Cu2+ and thiol compounds.

High-pressure system for Compton scattering experiments.

High-pressure apparatus for Compton scattering experiments has been developed to study the momentum distribution of conduction electrons in metals and alloys at high pressure. This apparatus was applied to observe the Compton profile of metallic Li under pressure. It was found that the Compton profile at high pressure could be obtained within several hours by using this apparatus and synchrotron radiation. The result on the pressure dependence of the Fermi momentum of Li obtained here is in good agreement with that predicted from the free-electron model.

Phase transitions of CdS microcrystals under high pressure.

The experiment under high pressure using a diamond anvil cell (DAC) requires the utilization of synchrotron radiation. High-pressure experiments were performed using a DAC on CdS microcrystals, both in the far-IR and in the X-ray regions, in order to study the lattice dynamics and lattice stability concerned with the phase transitions. From these experiments, experimental evidence is presented indicating that a CdS microcrystal of smaller diameter shows a higher transition pressure for lattice transformation under pressure. The origin of such an increase in the transition pressure in the microcrystals is discussed in relation to the surface tension.

Density measurements of liquid under high pressure and high temperature.

A new method for density measurements by means of X-ray absorption under high pressure and high temperature using synchrotron radiation has been developed. The method has been modified for a large-volume Paris-Edinburgh press and combined with intense high-energy X-rays at the ESRF. In order to overcome effects of deformation of sample shape under pressure, a ruby cylinder was used as a sample container. The density was determined from the intensity profile of transmitted X-rays. The densities of crystalline and liquid Bi were successfully measured up to 750 K at 1 GPa.

Membrane permeability of coelenterazine analogues measured with fish eggs.

To determine the suitability of various coelenterazine analogues for the regeneration of aequorin in living cells, the membrane permeabilities of 11 analogues were measured using the eggs of the killifish Fundulus grandis by soaking the eggs in solutions containing the analogues. The results indicated that e-coelenterazine, which has an exceptionally high rate of in vitro regeneration of aequorin, not only permeated poorly into the eggs but also was highly unstable. All other analogues tested permeated sufficiently into the eggs. The highest permeability was found with f-coelenterazine; the concentration of f-coelenterazine in the eggs was about five times that in the surrounding medium, assuming that the distribution of the compound in the egg is uniform.