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At least 379 records · Page 21

The Structure of the Statocyst of the Freshwater Snail Biomphalaria Glabrata (Pulmonata, Basommatophora)

The structure of the statocyst of the freshwater snail Biomphalaria glabrata has been examined by light and electron microscopy. The two statocysts are located on the dorsal-lateral side of the left and right pedal ganglion. The statocysts are spherical, fluid-filled capsules with a diameter of approximately 60 microns for young and 110 microns for adult snails. The wall of the cyst is composed of large receptor cells and many smaller supporting cells. The receptor cells bear cilia which are evenly distributed on the apical surface. The cilia have the typical 9+2 internal tubule configuration. Striate rootlets originate from the base of the basal body and run downward into the cytoplasm. Side-roots arise from one side of the basal body and a basal foot from the other. For each receptor cell, the basal foot always points to the periphery of the surface, indicating that the receptor cell is non-polarized. The receptor cells contain cytoplasmic organelles such as mitochondria, ribosomes, rough and smooth endoplasmic reticulum, compact Golgi bodies and multivesicular bodies. Supporting cells bearing microvilli are interposed between the receptor cells. The junction complex between the supporting cells and the receptor cells is composed of adherens and septate junctions, while between supporting cells only the adherens junctions are present. The static nerve arises from the lateral side of the cyst and contains axons in which parallel neurotubules and mitochondria are found. The axons arise directly from the base of the receptor cells without synapse. In the cyst lumen there are unattached statoconia. The statoconia have a plate-like or concentric membranous ring structure. Based on the morphology, the function of the statocyst in Biomphalaria is discussed.

Gao, Wenyuan↗

Modeling the Activity of Single Genes

The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. This picture is significantly augmentated when we consider the action of certain proteins in regulating transcription. These transcription factors provide a feedback pathway by which genes can regulate one another's expression as mRNA and then as protein. To review: DNA, RNA and proteins have different functions. DNA is the molecular storehouse of genetic information. When cells divide, the DNA is replicated, so that each daughter cell maintains the same genetic information as the mother cell. RNA acts as a go-between from DNA to proteins. Only a single copy of DNA is present, but multiple copies of the same piece of RNA may be present, allowing cells to make huge amounts of protein. In eukaryotes (organisms with a nucleus), DNA is found in the nucleus only. RNA is copied in the nucleus then translocates(moves) outside the nucleus, where it is transcribed into proteins. Along the way, the RNA may be spliced, i.e., may have pieces cut out. RNA then attaches to ribosomes and is translated to proteins. Proteins are the machinery of the cell other than DNA and RNA, all the complex molecules of the cell are proteins. Proteins are specialized machines, each of which fulfills its own task, which may be transporting oxygen, catalyzing reactions, or responding to extracellular signals, just to name a few. One of the more interesting functions a protein may have is binding directly or indirectly to DNA to perform transcriptional regulation, thus forming a closed feedback loop of gene regulation. The structure of DNA and the central dogma were understood in the 50s; in the early 80s it became possible to make arbitrary modifications to DNA and use cellular machinery to transcribe and translate the resulting genes; more recently, genomes (i.e., the complete DNA sequence) of many organisms have been sequenced. This large-scale sequencing began with simple organisms, viruses and bacteria, progressed to eukaryotes such as yeast, and more recently (1998) progressed to a multi-cellular animal, the nematode Caenorhabditis elegans. Sequencers have now moved on to the fruit fly Drosophila melanogaster, whose sequence is slated for completion by the end of 1999. The human genome project is expected to determine the complete sequence of all 3 billion bases of human DNA within the next five years. In the wake of genome-scale sequencing, further instrumentation is being developed to assay gene expression and function on a comparably large scale. Much of the work in computational biology focuses on computational tools used in sequencing, finding genes that are related to a particular gene, finding which parts of the DNA code for proteins and which do not, understanding what proteins will be formed from a given length of DNA, predicting how the proteins will fold from a one-dimensional structure into a three dimensional structure, and so on. Much less computational work has been done regarding the function of proteins. One reason for this is that different proteins function very differently, and so work on protein function is very specific to certain classes of proteins. There are, for example, proteins such enzymes that catalyze various intracellular reactions, receptors that respond to extracellular signals and ion channels that regulate the flow of charged particles into and out of the cell. In this chapter, we will consider a particular class of proteins called transcription factors(TFs), which are responsible for regulating when a certain gene is expressed in a certain cell, which cells it is express in, and how much is expressed. Understanding these processes will involve developing a deeper understanding of transcription, translation, and the cellular processes that control those processes. All of these elements fall under the aegis of gene regulation or more narrowly transcriptional regulation. Some of the key questions in gene regulation are: What genes are expressed in a certain cell at a certain time? How does gene expression differ from cell to cell in a multicellular organism? Which proteins act as transcription factors, i.e., are important in regulating gene expression? From questions like these, we hope to understand which genes are important for various macroscopic processes. Nearly all of the cells of a multicellular organism contain the same DNA. Yet this same genetic information yields a large number of different cell types. The fundamental difference between a neuron and a liver cell, for example, is which genes are expressed. Thus understanding gene regulation is an important step in understanding development. Furthermore, understanding the usual genes that are expressed in cells may give important clues about various diseases. Some diseases, such as sickle cell anemia and cystic fibrosis, are caused by defects in single, non-regulatory genes; others, such as certain cancers, are caused when the cellular control circuitry malfunctions - an understanding of these diseases will involve pathways of multiple interacting gene products. There are numerous challenges in the area of understanding and modeling gene regulation. First and foremost, biologists would like to develop a deeper understanding of the processes involved, including which genes and families of genes are important, how they interact, etc. From a computation point of view, there has been embarrassingly little work done. In this chapter there are many areas in which we can phrase meaningful, non-trivial computational questions, but questions that have not been addressed. Some of these are purely computational (what is a good algorithm for dealing with a model of type X) and others are more mathematical (given a system with certain characteristics, what sort of model can one use? How does one find biochemical parameters from system-level behavior using as few experiments as possible?). In addition to biological and algorithmic problems, there is also the ever-present issue of theoretical biology - what general principles can be derived from these systems, what can one do with models other than just simulate time-courses, what can be deduced about a class of systems without knowing all the details? The fundamental challenge to computationalists and theorists is to add value to the biology - to use models, modeling techniques and algorithms to understand the biology in new ways.

Mjolsness, Eric↗

Thermococcus sulfurophilus sp. nov., a New Hyperthermophilic, Sulfur-Reducing Archaeon Isolated from Deep-Sea Hydrothermal Vent

A new hyperthermophilic, anaerobic, sulfur-reducing, organo-heterotrophic archaeon, strain OGL-20P, was isolated from "black smoker" chimney material at the Rainbow hydrothermal vent site in the Atlantic Ocean (36.2 N; 33.9 W). The cells of strain OGL-20P have irregular coccoid shape and are motile with a single flagellum. Growth occurs within pH range of 5.5-8.2 (optimal at pH 7.0-7.2), salinity range of 1-5% NaCl (optimal concentration 3% NaCl wt/vol), and temperature range of +55 C to +94 C (optimal growth at +83 C to +85 C). Strain OGL-20P is resistant to freezing (at -20 C). New isolate is strictly anaerobic with sulfur-type of respiration. A limited number of compounds are utilized as electron donors, including peptone, becto-tryptone, casamino-acids, and yeast extract but does not grow with separate amino acids. Sulfur and Iron can be used as electron acceptors; but not sulfate, sulfite, thiosulfate or nitrate. Strain OGL-20P is resistant to chloramphenicol, kanamycin, and gentamycin. Growth of str. OGL20P is inhibited by tetracyclin but not by Na2MoO4. The G+C content of DNA is 57.2 mol%. The 16S ribosomal RNA sequence analysis allows one to classify strain OGL-20P as a representative of a now species of Thermococcus genus. The name Thermococcus sulfurophilus op. nov., was suggested for the new isolate, type strain OGL-20P (sup T) (= ATCC BAA_394 (sup T) = DSM...(supT)).

Pikuta, Elena V.↗

Microbial identification by immunohybridization assay of artificial RNA labels

Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA-RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA-RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.

Non-NASA Center↗

Arthromitus (Bacillus cereus) symbionts in the cockroach Blaberus giganteus: dietary influences on bacterial development and population density

The filamentous spore-forming bacterium Arthromitus, discovered in termites, millipedes, sow bugs and other soil-dwelling arthropods by Leidy (1850), is the intestinal stage of Bacillus cereus. We extend the range of Arthromitus habitats to include the hindgut of Blaberus giganteus, the large tropical American cockroach. The occurrence and morphology of the intestinal form of the bacillus were compared in individual cockroaches (n=24) placed on four different diet regimes: diurnally maintained insects fed (1) dog food, (2) soy protein only, (3)purified cellulose only, and (4) a dog food-fed group maintained in continuous darkness. Food quality exerted strong influence on population densities and developmental stages of the filamentous bacterium and on fecal pellet composition. The most dramatic rise in Arthromitus populations, defined as the spore-forming filament intestinal stage, occurred in adult cockroaches kept in the dark on a dog food diet. Limited intake of cellulose or protein alone reduced both the frequency of Arthromitus filaments and the rate of weight gain of the insects. Spores isolated from termites, sow bugs, cockroaches and moths, grown on various hard surfaces display a branching mobility and resistance to antibiotics characteristic to group I Bacilli whose members include B. cereus, B. circulans, B. alvei and B. macerans. DNA isolated from pure cultures of these bacilli taken from the guts of Blaberus giganteus (cockroach), Junonia coenia (moth), Porcellio scaber (sow bug) and Cryptotermes brevis (termite) and subjected to Southern hybridization with a 23S-5S B. subtilis ribosomal sequence probe verified that they are indistinguishable from laboratory strains of Bacillus cereus.

NASA Discipline Exobiology↗

Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach

The utility of a high-density oligonucleotide microarray (microchip) for identifying strains of five closely related bacilli (Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus medusa and Bacillus subtilis) was demonstrated using an approach that compares the non-equilibrium dissociation rates ('melting curves') of all probe-target duplexes simultaneously. For this study, a hierarchical set of 30 oligonucleotide probes targeting the 16S ribosomal RNA of these bacilli at multiple levels of specificity (approximate taxonomic ranks of domain, kingdom, order, genus and species) was designed and immobilized in a high-density matrix of gel pads on a glass slide. Reproducible melting curves for probes with different levels of specificity were obtained using an optimized salt concentration. Clear discrimination between perfect match (PM) and mismatch (MM) duplexes was achieved. By normalizing the signals to an internal standard (a universal probe), a more than twofold discrimination (> 2.4x) was achieved between PM and 1-MM duplexes at the dissociation temperature at which 50% of the probe-target duplexes remained intact. This provided excellent differentiation among representatives of different Bacillus species, both individually and in mixtures of two or three. The overall pattern of hybridization derived from this hierarchical probe set also provided a clear 'chip fingerprint' for each of these closely related Bacillus species.

Non-NASA Center↗

Metabolism of proteinoid microspheres

The literature of metabolism in proteinoids and proteinoid microspheres is reviewed and criticized from a biochemical and experimental point of view. Closely related literature is also reviewed in order to understand the function of proteinoids and proteinoid microspheres. Proteinoids or proteinoid microspheres have many activities. Esterolyis, decarboxylation, amination, deamination, and oxidoreduction are catabolic enzyme activities. The formation of ATP, peptides or oligonucleotides is synthetic enzyme activities. Additional activities are hormonal and inhibitory. Selective formation of peptides is an activity of nucleoproteinoid microspheres; these are a model for ribosomes. Mechanisms of peptide and oligonucleotide syntheses from amino acids and nucleotide triphosphate by proteinoid microspheres are tentatively proposed as an integrative consequence of reviewing the literature.

Non-NASA Center↗

Metabolism of proteinoid microspheres

The literature of metabolism in proteinoids and proteinoid microspheres is reviewed and criticized from a biochemical and experimental point of view. Closely related literature is also reviewed in order to understand the function of proteinoids and proteinoid microspheres. Proteinoids or proteinoid microspheres have many activities. Esterolysis, decarboxylation, amination, deamination, and oxidoreduction are catabolic enzyme activities. The formation of ATP, peptides or oligonucleotides is synthetic enzyme activities. Additional activities are hormonal and inhibitory. Selective formation of peptides is an activity of nucleoproteinoid microspheres; these are a model for ribosomes. Mechanisms of peptide and oligonucleotide syntheses from amino acids and nucleotide triphosphate by proteinoid microspheres are tentatively proposed as an integrative consequence of reviewing the literature.

Non-NASA Center↗

A low molecular weight artificial RNA of unique size with multiple probe target regions

Artificial RNAs (aRNAs) containing novel sequence segments embedded in a deletion mutant of Vibrio proteolyticus 5S rRNA have previously been shown to be expressed from a plasmid borne growth rate regulated promoter in E. coli. These aRNAs accumulate to high levels and their detection is a promising tool for studies in molecular microbial ecology and in environmental monitoring. Herein a new construct is described which illustrates the versatility of detection that is possible with aRNAs. This 3xPen aRNA construct carries a 72 nucleotide insert with three copies of a unique 17 base probe target sequence. This aRNA is 160 nucleotides in length and again accumulates to high levels in the E. coli cytoplasm without incorporating into ribosomes. The 3xPen aRNA illustrates two improvements in detection. First, by appropriate selection of insert size, we obtained an aRNA which provides a unique and hence, easily quantifiable peak, on a high resolution gel profile of low molecular weight RNAs. Second, the existence of multiple probe targets results in a nearly commensurate increase in signal when detection is by hybridization. These aRNAs are naturally amplified and carry sequence segments that are not found in known rRNA sequences. It thus may be possible to detect them directly. An experimental step involving RT-PCR or PCR amplification of the gene could therefore be avoided.

Non-NASA Center↗

Cytology of long-term desiccation in the desert cyanobacterium Chroococcidiopsis (Chroococcales)

Young and old cultures (up to 66 months) of two Chroococcidiopsis sp. strains isolated from the Negev desert, Israel, were examined by epifluorescence and electron microscopy. In old cultures, cell viability and autofluorescence were lower than in young cultures. An increase was seen with age in the polysaccharide content of the sheaths of nanocytes and nanocyte mother cells, and a decrease of phycobiliproteins was also seen. In the oldest cultures most of the cells were dead and in various stages of degeneration. Single living cells were scattered among the dead ones. No resting cells were formed in the oldest cultures, but many cell groups showed highly electron-dense sheaths and, in the cytoplasm, ribosomes and glycogen. These changes in cell structure may have a role in preventing water loss from the cell.

NASA Discipline Exobiology↗

Partial purification and characterization of a Ca(2+)-dependent protein kinase from pea nuclei

Almost all the Ca(2+)-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca2+ and reaches half-maximal activation at about 3 x 10(-7) molar free Ca2+, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca(2+)-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca(2+)-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.

NASA Discipline Number 40-50↗

Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins

Features of the physical environment surrounding an ancestral organism can be inferred by reconstructing sequences of ancient proteins made by those organisms, resurrecting these proteins in the laboratory, and measuring their properties. Here, we resurrect candidate sequences for elongation factors of the Tu family (EF-Tu) found at ancient nodes in the bacterial evolutionary tree, and measure their activities as a function of temperature. The ancient EF-Tu proteins have temperature optima of 55-65 degrees C. This value seems to be robust with respect to uncertainties in the ancestral reconstruction. This suggests that the ancient bacteria that hosted these particular genes were thermophiles, and neither hyperthermophiles nor mesophiles. This conclusion can be compared and contrasted with inferences drawn from an analysis of the lengths of branches in trees joining proteins from contemporary bacteria, the distribution of thermophily in derived bacterial lineages, the inferred G + C content of ancient ribosomal RNA, and the geological record combined with assumptions concerning molecular clocks. The study illustrates the use of experimental palaeobiochemistry and assumptions about deep phylogenetic relationships between bacteria to explore the character of ancient life.

Bacteria/classification/metabolism↗

Macromolecular differentiation of Golgi stacks in root tips of Arabidopsis and Nicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples

The plant root tip represents a fascinating model system for studying changes in Golgi stack architecture associated with the developmental progression of meristematic cells to gravity sensing columella cells, and finally to "young" and "old", polysaccharide-slime secreting peripheral cells. To this end we have used high pressure freezing in conjunction with freeze-substitution techniques to follow developmental changes in the macromolecular organization of Golgi stacks in root tips of Arabidopsis and Nicotiana. Due to the much improved structural preservation of all cells under investigation, our electron micrographs reveal both several novel structural features common to all Golgi stacks, as well as characteristic differences in morphology between Golgi stacks of different cell types. Common to all Golgi stacks are clear and discrete differences in staining patterns and width of cis, medial and trans cisternae. Cis cisternae have the widest lumina (approximately 30 nm) and are the least stained. Medial cisternae are narrower (approximately 20 nm) and filled with more darkly staining products. Most trans cisternae possess a completely collapsed lumen in their central domain, giving rise to a 4-6 nm wide dark line in cross-sectional views. Numerous vesicles associated with the cisternal margins carry a non-clathrin type of coat. A trans Golgi network with clathrin coated vesicles is associated with all Golgi stacks except those of old peripheral cells. It is easily distinguished from trans cisternae by its blebbing morphology and staining pattern. The zone of ribosome exclusion includes both the Golgi stack and the trans Golgi network. Intercisternal elements are located exclusively between trans cisternae of columella and peripheral cells, but not meristematic cells. In older peripheral cells only trans cisternae exhibit slime-related staining. Golgi stacks possessing intercisternal elements also contain parallel rows of freeze-fracture particles in their trans cisternal membranes. We propose that intercisternal elements serve as anchors of enzyme complexes involved in the synthesis of polysaccharide slime molecules to prevent the complexes from being dragged into the forming secretory vesicles by the very large slime molecules. In addition, we draw attention to the similarities in composition and apparent site of synthesis of xyloglucans and slime molecules.

NASA Program Space Biology↗

Modulation of statolith mass and grouping in white clover (Trifolium repens) growth in 1-g, microgravity and on the clinostat

Current models of gravity perception in higher plants focus on the buoyant weight of starch-filled amyloplasts as the initial gravity signal susceptor (statolith). However, no tests have yet determined if statolith mass is regulated to increase or decrease gravity stimulus to the plant. To this end, the root caps of white clover (Trifolium repens) grown in three gravity environments with three different levels of gravity stimulation have been examined: (i) 1-g control with normal static gravistimulation, (ii) on a slow clinostat with constant gravistimulation, and (iii) in the stimulus-free microgravity aboard the Space Shuttle. Seedlings were germinated and grown in the BioServe Fluid Processing Apparatus and root cap structure was examined at both light and electron microscopic levels, including three-dimensional cell reconstruction from serial sections. Quantitative analysis of the electron micrographs demonstrated that the starch content of amyloplasts varied with seedling age but not gravity condition. It was also discovered that, unlike in starch storage amyloplasts, all of the starch granules of statolith amyloplasts were encompassed by a fine filamentous, ribosome-excluding matrix. From light micrographic 3-D cell reconstructions, the absolute volume, number, and positional relationships between amyloplasts showed (i) that individual amyloplast volume increased in microgravity but remained constant in seedlings grown for up to three days on the clinostat, (ii) the number of amyloplasts per cell remained unchanged in microgravity but decreased on the clinostat, and (iii) the three-dimensional positions of amyloplasts were not random. Instead amyloplasts in microgravity were grouped near the cell centers while those from the clinostat appeared more dispersed. Taken together, these observations suggest that changing gravity stimulation can elicit feedback control over statolith mass by changing the size, number, and grouping of amyloplasts. These results support the starch-statolith theory of graviperception in higher plants and add to current models with a new feedback control loop as a mechanism for modulation of statolith responsiveness to inertial acceleration.

Flight Experiment↗

Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation

The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis. Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.

NASA Program Fundamental Space Biology↗

Cosmopolitan distribution of the large composite microbial mat spirochete, Spirosymplokos deltaeiberi

Inocula from organic-rich black muds immediately underlying intertidal laminated microbial mats dominated by Microcoleus chthonoplastes yielded large, variable diameter spirochetes. These unusual spirochetes, previously reported only from the Alfacs Peninsula at the delta of the Ebro river in northeast Spain, contain striking arrays of cytoplasmic granules packed into their protoplasmic cylinders. On several occasions, both in summer and winter, the huge spirochetes were recognized in samples from mats growing in the Sippewissett salt marsh at Woods Hole Massachusetts. They were also seen in similar samples from microbial mats at North Pond, Laguna Figueroa, Baja California Norte, Mexico. The identity of these spirochetes was confirmed by electron microscopy: number and disposition of flagella, composite structure, measurements of their distinctive cytoplasmic granules. The granules, larger, more conspicuous and present in addition to ribosomes, are hypothesized to contain ATPases. As culture conditions worsen, these spirochetes retract into membrane-bounded round bodies in which they form refractile inclusions. From morphology and behavior we conclude the North American spirochetes from both Atlantic and Pacific intertidal microbial mats are indistinguishable from those at the delta of the Ebro river. We conclude a cosmopolitan distribution for Spirosymplokos deltaeiberi.

NASA Discipline Exobiology↗

Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region

Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.

NASA Discipline Regulatory Physiology↗

Production of otoconia in the endolymphatic sac in the Japanese red-bellied newt, Cynops pyrrhogaster: light and transmission electron microscopic study

The formation of otoconia in the endolymphatic sac (ES) of the larval newt, Cynops pyrrhogaster, has been studied by light and transmission electron microscopy. Some of the epithelial cells of the ES contain an abundance of swollen vesicles, Golgi complexes, rough endoplasmic reticula and ribosomes at the late larval stages 50 and 51, approximately 26-30 days after eggs are laid. Five days later, at stage 52, crystals are present in the vacuoles between the epithelial cells. Serial sections indicate that these vacuoles actually form small canals which lie in the wall and join the lumen of the ES. Reconstruction of the ES shows that several canals are contained in the ES wall. At stage 56, about 72 days after eggs are laid, a large number of otoconia are present in the ES lumen, while the otoconia disappear from the canals. It appears that the otoconia are first produced in the canals and then released to the lumen. Some epithelial cells of the ES are thought to expel the organic and inorganic material to the canals to form the otoconia in situ. The process of formation of the otoconia in the ES is discussed.

Non-NASA Center↗