Unexpected diversity of bioluminescence in planktonic worms.

The vast majority of pelagic bioluminescent organisms emit a blue light with emission maxima (λmax ) ranging from 450 to 490 nm. Among the known outliers, the tomopterids (Annelida: Polychaeta) are usually described as yellow-emitters (λmax  = 565-570 nm) for which bioluminescence functions as a specific recognition signal. Here, we report the first data regarding the colours emitted by four different tomopterid species, Tomopteris pacifica, T. carpenteri, T. septentrionalis and T. planktonis. Surprisingly, T. planktonis is a blue-emitter (λmax  = 450 nm). Our pharmacological results on T. planktonis support cholinergic control, as recently demonstrated in the yellow-emitter, T. helgolandica. Moreover, as revealed by epifluorescence microscopy, the light seems to be produced in both species from the same yellow-pigmented parapodial glands. Despite these similarities, tomopterids express an unexpected diversity of bioluminescent colour patterns. This leads us to reassess the ecological value of bioluminescence within this group.

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.

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.

Genetic relationship of putative colonization factor O166 to colonization factor antigen I and coli surface antigen 4 of enterotoxigenic Escherichia coli.

Plasmid DNA from two strains of enterotoxigenic Escherichia coli harboring genes encoding coli surface antigen 4 (CS4) and from seven Indian enterotoxigenic E. coli isolates cross-hybridized at low stringency but not at high stringency with two polynucleotide probes derived from the colonization factor antigen I (CFA/I) operon. Low-stringency Southern blot hybridization of PstI-digested plasmid DNA from the seven Indian isolates yielded characteristic restriction fragment patterns, distinct from those of CS4- and CFA/I-associated plasmid DNA. Two of the Indian strains were transformed with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 genes. The cfaD transformants produced large amounts of putative colonization factor O166 (PCFO166) irrespective of whether the nutrient agar contained bile salts, a growth factor otherwise required for adequate PCFO166 expression. A considerable interstrain variation in the level of PCFO166 production could be explained by differences in the proportion of bacteria that were fimbriated, as visualized by electron microscopy. The N-terminal amino acid sequence of PCFO166 fimbrial protein showed a high degree of homology with the corresponding sequences of CFA/I and CS4.

Myasthenia gravis patients with a thymoma have antibodies against a high molecular weight protein in sarcoplasmic reticulum.

Our purpose was to investigate whether components of the sarcoplasmic reticulum (SR) are relevant antigens in myasthenia gravis (MG). Using enzyme-linked immunosorbent assay (ELISA), 75 MG sera and 120 control sera were examined for IgG antibodies against SR prepared from rabbit skeletal muscle. 16/30 thymoma MG patients had IgG antibodies that reacted with SR. 1/30 MG patients with thymic hyperplasia and 3/15 MG patients with thymic atrophy had SR antibodies in low concentrations. Control sera were negative. Using immunoblot, SR antibodies were detected in the thymoma group only. 14/30 sera from thymoma patients reacted with a protein of 320 kDa relative molecular weight. The only reported SR protein with similar electrophoretic mobility is the subunit of the spanning protein which links junctional SR to sarcolemma and functions as a calcium-release channel.

Altered ciliary substructure in the endosalpinx in women using an IUCD.

Fallopian tube segments were removed from 20 women undergoing sterilization by laparoscopy or laparotomy. Ten of these patients used an intrauterine contraceptive device (IUCD). The other 10 had used neither IUCD nor oral contraceptives and served as controls. The ciliary ultrastructure was examined by light- and transmission electron microscopy. The IUCD users had a significantly reduced ciliary length and less well oriented cilia, as compared with the control group. Also, the proportion of cilia with a ciliary crown structure was significantly smaller in IUCD users than in the non-users. The mechanism that may cause these alterations and their putative consequences are discussed.

Visualization of the Transparent, Gelatinous House of the Pelagic Tunicate Oikopleura vanhoeffeni Using Sepia Ink.

Appendicularian tunicates of the genus Oikopleura feed using an external, acellular, transparent structure known as the house. Previously, dilute particulate dyes have been used to visualize the internal structure of this house. However, because of toxicity, large particle size, and flocculation, many of these dyes have been of limited practical and scientific use. We report on a new marker, the ink from the cephalopod Sepia officinalis, that solves many of these problems. Specimens of Oikopleura vanhoeffeni relished Sepia ink, having dark black stomachs and producing many dark fecal pellets over several days. When O. vanhoeffeni expanded houses in dilute ink, the internal walls, septae, and filters were shown in great detail, whereas high concentrations of ink showed delicate patterns of lines on the internal walls. We present documentary photographs of previously unillustrated or undescribed morphologies: the escape slot; the incurrent funnels; two dimples caused by insertion of suspensory filaments on the upper wall of the posterior chamber, a large, posterior keel; both the open and closed positions of the exit valve; and the complex pattern of lines on the inner walls. However, the external walls of the house had no affinity for the dye and could only be seen by dark field illumination. We believe that Sepia ink can be used to visualize functionally important transparent structures of other gelatinous zooplankton and can be a colloidal marker in feeding experiments of a wide range of filter feeders.

Pharmacological manipulation with the descending serotonergic system or transection of the mouse spinal cord has no effect on ependymal ultrastructure.

In order to investigate an effect of descending nerve fibres on mouse spinal cord ependymal ultrastructure, pharmacological manipulation with the serotonergic system or transection of the spinal cord was done. Biochemical analysis showed an 83% reduction of serotonin content in spinal cord tissue after p-chlorophenylalanine injections and a 93% reduction after transection. However, none of the experimental animals showed changes in ependymal ultrastructure compared to control animals as revealed by electron microscopy.

Ultrastructure of the mouse spinal cord ependyma.

This study was done in order to investigate the normal ultrastructure of well-preserved mouse spinal canal ependyma using light, scanning and transmission electron microscopy. The ependymal lining was found to consist of a simple, cuboidal epithelium essentially similar to the unspecialized cuboidal ependyma of the brain ventricles. Apart from great variation in kinociliary density, no intracellular difference was noted between the ependymal cells. In contrast to earlier findings, indications of the existence of zonulae occludentes between the apical part of the ependymal cells were observed. Our findings do not support the hypothesis of secretion or intracellular transport by the ependyma, or that the ependyma constitutes a significant diffusion barrier.

Quinolinic acid metabolism in the rat brain. Immunohistochemical identification of 3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase in the hippocampal region.

Quinolinic acid (QUIN) is a potent endogenous excitotoxin, which has been shown to be present in the brain (Wolfensberger et al., 1983). In order to study the cellular localization of QUIN metabolism in the hippocampus, specific antibodies raised against purified rat liver 3-hydroxyanthranilic acid oxygenase (3HAO) and quinolinic acid phosphoribosyltransferase (QPRT), the enzymes directly responsible for QUIN synthesis and catabolism, respectively, were used for immunohistochemical studies in the adult male rat. Cells containing 3HAO immunoreactivity (3HAO-i) were present in all subfields of the hippocampal region, including the area dentata, Ammon's horn, the subicular complex, and the entorhinal area. The highest density of 3HAO-i cells was found in the molecular layer of Ammon's horn and in the hilus of area dentata, while the granular cell layer of area dentata and stratum pyramidale of Ammon's horn contained the lowest number of 3HAO-stained cells. A majority of hippocampal 3HAO-i cells were also stained with monoclonal antibodies against glial fibrillary acidic protein (GFAP) or S-100 protein, suggesting that 3HAO-i is present primarily in astrocytes. At the ultrastructural level, 3HAO-i was found to be distributed uniformly throughout the cytoplasm, with intense immunostaining present in the internal and the external layers of the mitochondria. QPRT-i was detected in 3 morphologically distinct cell types present in all parts of the hippocampus. The total number of QPRT-i cells was lower than that of the 3HAO-i cells. QPRT-i cells were relatively numerous in the molecular and radial layers of Ammon's horn, while they occurred only sporadically in stratum pyramidale of Ammon's horn and in the granular cell layer of area dentata. Many QPRT-i cells stained with antibodies against GFAP and S-100, but the proportion of cells in which QPRT was colocalized with these glial marker proteins was lower than that for 3-HAO-i cells. At the ultrastructural level, 2 types of QPRT-i glial cells were detected. The smaller cell type had a diffuse cytoplasmic staining, while the larger cell type, which also contained glial filaments, showed diffuse cytoplasmic staining and intense staining of lysosomal structures. The observation that 3HAO and QPRT only partially coexist in hippocampal glial cells suggests that while synthesis and catabolism of QUIN may occur in the same glial cells, catabolism of QUIN can also take place in cells lacking the synthetic enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Preservation of the ependyma of the mouse spinal cord by various vascular perfusion procedures.

Seventy-two mice were used to find out which of 13 vascular perfusion procedures gave best structural preservation of the spinal cord ependyma and central canal lumen. Best results were obtained by 3% glutaraldehyde in Tyrodes solution with 50% of normal NaCl amount, 0.06 M sucrose and 2% dextran T-40 (556 mOsmol, pH 7.2, 0-4 degrees C). This was perfused by a peristaltic pump at 40 ml/min for 10 min through a cannula inserted in the ascending aorta. No advantage was seen by heparin pretreatment or adding a prefixation rinse. With good tissue preservation the central canal was found to be round to oval in cross-sectional profile and almost free of intraluminal material.

Effects of dextran-containing perfusion fixatives on intraluminal compounds in rat kidney tubules.

In the present study on rat kidney tubules an intraluminal, electron-dense granular substance was found in animals perfusion-fixed with dextran-containing fluids. Most findings were made in thick ascending limbs of Henle and distal convoluted tubules. The incidence decreased from 50% to zero when intravascular rinsing time was increased from zero to 70 sec. In animals fixed without dextran no such substance was found. In some thick ascending limbs of Henle and distal convoluted tubules a similar electron-dense substance was found along the apical plasmalemma of some cells. The origin of the observed substances is at present not defined, but may represent a coprecipitation of dextran and (glyco)proteins caused by glutaraldehyde.

Scanning electron microscopy of human urine and purified Tamm-Horsfall's glycoprotein.

Dialyzed and dehydrated human urine and purified Tamm-Horsfall's glycoprotein revealed similar morphology by scanning electron microscopy. Single filaments, with diameters between 15-45 nm, splitting off and merging with thicker fibers at irregular intervals made up a three-dimensional meshwork with submicrometer pores. The resulting "fishing net" is capable of trapping microorganisms and may facilitate their elimination from the urinary tract by micturition. Tamm-Horsfall's glycoprotein may accordingly be a factor protecting against urinary tract infections.

The distribution of lead in human hemopoietic tissue and spongy bone after lead poisoning and Ca-EDTA chelation therapy. Observations made by atomic absorption spectroscopy, laser microbeam mass analysis and electron microbeam X-ray analysis.

Two iliac crest needle biopsies were taken from a 43-year-old lead-poisoned woman during and after completion of a Ca-EDTA treatment. By atomic absorption spectroscopy the first and second biopsy were found to contain 56, respectively 41.6 micrograms lead/g wet tissue. In both biopsies 36% of the lead was extractable in 0.1 N HCl. Electron microbeam X-ray analysis proved to have too low sensitivity for quantitation of the lead in these biopsies. Laser microbeam mass analysis (LAMMA), performed only on the second biopsy, revealed a high and fairly constant residual lead concentration in all bone marrow cell nuclei (approximately 55 micrograms/g) and a low lead concentration in the cytoplasm of the same cells (4-12 micrograms/g). The extracellular bone matrix lead was greatly concentrated in the superficial 3-6 microns osteoid zone of the bony trabeculae and totally absent from deeper parts of the mineralized matrix. The LAMMA results are in good agreement with those of subcellular fractionation experiments and atomic absorption spectroscopy, provided that the relative volume fraction of nucleus and cytoplasm is accounted for. The high residual osteoid lead after completed chelation therapy indicates that lead has a stronger affinity for the organic than the mineral components of bone matrix.

Quinolinic acid phosphoribosyltransferase: preferential glial localization in the rat brain visualized by immunocytochemistry.

The excitotoxic brain metabolite quinolinic acid has been hypothetically linked to the pathogenesis of neurodegenerative disorders. By using antibodies prepared against a homogeneous preparation of its catabolic enzyme, quinolinic acid phosphoribosyltransferase [QPRTase; nicotinate-nucleotide:pyrophosphate phosphoribosyltransferase (carboxylating), EC 2.4.2.19], immunocytochemical methods were applied to assess the cellular and subcellular localization of quinolinic acid in the rat brain. On the light-microscopic level, the enzyme was found to be preferentially associated with glial elements of variable morphology. In addition to its presence in glial cells, QPRTase was contained in tanycytes and ependymal cells of the cerebral ventricles and, sporadically, in neurons. Overall, QPRTase immunoreactivity was noted in every brain region studied, the histological pattern being in good accordance with the regional variation of enzyme activity established in biochemical studies. As judged on the ultrastructural level, QPRTase, in all cell types examined so far, was often noted in densely stained roundish cytoplasmic bodies (0.1-0.8 micron in diameter), which were bounded by a single membrane. In functional terms, these structures may represent early lysosomes, secretory granules, or residual bodies. The particular anatomical arrangement of the quinolinic acid system may reflect the brain's defense strategy against detrimental effects of the endogenous excitotoxin.

Mast cells in the tubal wall in women using an intrauterine contraceptive device.

The number of mast cells in the tubal wall of 33 healthy nonpregnant women, 17 of whom had an intrauterine contraceptive device (IUCD), was investigated. Light microscopy showed that both the muscularis externa and the lamina propria of the tubal wall contained more mast cells in the 17 IUCD users than in the 16 non-users (control group). In both patient groups the mast cell concentration was higher in the muscularis externa than in the lamina propria. Most mast cells of the muscularis externa were more closely related to smooth muscle cells than to blood vessels. The increased number of mast cells in IUCD users may be a factor in the pathogenesis of pelvic inflammatory disease and the ectopic pregnancies that occur in IUCD users.

Immunohistochemical properties of the "floor plate glycogen body" of the human embryonic spinal cord and brain stem.

Prominent glycogen accumulations have been found in the floor plate radial glial cells of the human spinal cord and brain stem during the 6th to 13th week of intrauterine life. These glycogen-rich cells are totally negative to indirect immunoperoxidase staining with an antibody to glial fibrillary acidic protein, a protein that is strongly expressed in the remaining radial glial cells that border the central canal of the spinal cord. The glycogen-rich floor plate radial glial cells are, on the other hand, heavily stained by a monoclonal antibody against a vimentin-related protein. The neighbouring lateral radial glial cells do not express this protein. These and other distinctive features of the floor plate radial glial cells indicate an organoid specialisation of the floor plate during limited periods of intrauterine life. The function(s) of this specialised tissue remains obscure, but it may be related to cortico-spinal fibres crossing the midline through the floor plate, or to the transport of substances in both directions between blood vessels and the central canal.

Morphological changes in tubal mucosa associated with the use of intrauterine contraceptive devices.

We examined the endosalpinx of 28 healthy non-pregnant women, 12 of whom had an intrauterine contraceptive device (IUCD) and 16 of whom had neither an IUCD nor used oral contraceptives. Measurements made on light- and scanning electron micrographs showed that the percentage area covered by ciliated cells was approximately 40% in non-IUCD users and only 20% in IUCD users.

PIP: Given that IUD users have a high risk of ecotopic pregnancy and since most of these pregnancies are tubal, a study was undertaken in Norway to determine if there were any differences in the tubal epithelium of IUD users and women who never used IUDs. The proportion of the epithelium surface covered by ciliated cells was the major focus of the investigation. 1 cm segments from the isthmic section of the oviduct were removed from 12 IUD users and 16 nonusers while they were undergoing sterilization procedures. The women were all healthy and nonpregnant. Both groups of women were similar in terms of average age and parity. The IUDs used by the women included 5 copper Ts, 4 Multiloads, 1 Lippes Loop, and 2 IUDs of unknown type. All of the users had worn the devices for at least 6 months. The specimens of epithelium were immersed in a fixative and stained. The proportion of the epithetial surface covered by ciliated cells was measured using both light and scanning electron micrographs. The results obtained from the 2 methods of measuring differed somewhat for individual specimens; however, the overall results obtained from both methods were similar. Light microscropy measurements indicated that the proportion of epithetial surface covered by ciliated cells was 39.9% for nonuser and 21.2% for IUD users. Respective results obtained by scanning electron microscopy were 38.8% and 20.0%. The results were obtained from women in different menstrual cycle phases. A comparison of specimens by menstrual cycle phase revealed that the proportion of surface covered by ciliated cells was not affected by hormonal changes during the menstrual cycle. IUD users had a slightly higher intraepithelial leucocyte count than the control group. Whether or not reduced ciliation in IUD users plays a role in tubal pregnancy is not known; however, egg transport might be slowed down when there are fewer ciliated cells. Reduced ciliation might also affect sperm migration which in turn might enhance the risk of abnormal embryos. Most tubal pregnancies are characterized by abnormal embryos.

Phenoxyethanol as a nontoxic substitute for formaldehyde in long-term preservation of human anatomical specimens for dissection and demonstration purposes.

Formaldehyde has recently been declared a potential carcinogen. Occupational health authorities throughout the world are therefore likely to put stricter regulations to its use also within anatomical disciplines. We have been able to reduce the atmospheric concentration of formaldehyde in our dissection rooms to below the detection limit of a conventional Dräger tube multigas analyzer (i.e., below 0.5 ppm or 0.6 mg formaldehyde/m3 air), by extracting previously formaldehyde-fixed material for more than 3 months in 1% phenoxyethanol in tap water. In this fluid our material has remained soft and flexible with a consistency and color retention suitable for dissection and demonstration purposes for up to 10 years. Fungal attacks are rare and we have been unable to raise bacteria from such specimens. Even the microscopical structure of most tissues remains satisfactory after 5 years in 1% phenoxyethanol. The unpleasant and irritating smell traditionally felt in dissection rooms is almost absent in our facilities, but some of our students still mention slight odor, headache, drowsiness, and mild eye, nose, and throat irritation during their dissection practice periods.