bluuebunny/crossref_metadata_2025 huggingface dataset:
Emerging cell and molecular strategies for the study and treatment of painful peripheral neuropathies
Authors: Eaton Mary J.
Published: March 2003

Summary  Pharmacologic treatment for the symptoms of painful neuropathy has been problematic, because there has been limited understanding of the underlying etiologies and systemic levels that an effective dose can have on multiple side effects. The use of molecular methods, such as gene deletion from knockout mice and cellular minipumps for delivery of biologic antinociceptive molecules, has led to a better understanding of the underlying mechanisms of the induction of intractable neuropathic pain. The initiation of an excitatory cascade after injury or disease leads to the induction of various second messenger systems, loss or down‐regulation of the endogenous inhibitory spinal GABA system and central sensitization, causing such pain. The development and use of cellular minipumps, immortalized cell lines bioengineered to secrete various antinociceptive molecules for the reversal of neuropathic pain, makes cellular therapy a strategy for clinical use in the next few years. The development of molecular ”disimmortalization” technologies will make the use of such engineered cell lines safe for human use. Direct somatic gene transfer for neuropathic pain will eventually overcome the problems associated with transplantation of non‐autologous and xenogenic cells. These virus‐mediated methods, although at the early stages of evolution and use, offer large‐scale production of biologic agents that can be conveniently and confidently used for the long‐term relief of chronic neuropathic pain in a clinical setting, without systemic effects or surgical interventions.
Anterograde TNFα Transport From Rat Dorsal Root Ganglion To Spinal Cord And Injured Sciatic Nerve
Authors: Shubayev VI , Myers RR.
Published: March 2003

Tumor necrosis factor‐α (TNFα) is a key modulator of painful peripheral nerve injury. We have previously shown that a tracer of TNFα injected at the site of rat sciatic nerve injury undergoes retrograde axonal transport to the dorsal root ganglia (DRG). To further understand the role of TNFα in DRG, we injected rat L5 DRG with biotinylated TNFα, neurobiotin, or vehicle, and detected translocation of the biotin tag by avidin‐biotin histochemistry. Biotinylated TNFα was transported intraaxonally toward the periphery of both normal and injured nerves. It also reached the dorsal horn of the spinal cord in injured rats, but not in control rats. These findings highlight a dynamic process of TNFα axonal transport in the peripheral neural axis, and help explain activation of central cytokines in the pathogenesis of painful neuropathy.
GCPII (NAALADase) Inhibition Prevents Long‐Term Diabetic Neuropathy In Type 1 Diabetic BB/WOR Rats
Authors: Zhang W , Slusher B , Murakawa Y , Wozniak KM , Tsukamoto T , Jackson PF , Sima AAF.
Published: March 2003

Aims/Hypothesis: Hyperglutamatergic activity induced by ischemia is believed to underlie neuronal damage in a variety of neurological disorders, including neuropathic pain. Since ischemia is believed to be a prominent mechanism involved in diabetic polyneuropathy (DPN), we investigated the effect of the glutamate carboxypeptidase II (GCPII, EC #3.4.17.21; previously termed NAALADase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG to NAA and glutamate, on the development of DPN in type 1 diabetic BB/Wor rats. Methods: Diabetic animals were treated with 10 mg/kg/day i.p. of the selective GCPII inhibitor GPI‐5232 from onset of diabetes for 6 months. Hyperalgesia to thermal stimulation and nerve conduction velocity (NCV)) were measured monthly. The effect on structural DPN was assessed by scoring of single, teased myelinated fibers, myelinated fiber morphometry and ultrastructural examination of C‐fibers at 6 months. Results: GCPII inhibition showed significant but partial effects on hyperalgesia (p<0.001), nerve conduction slowing (p<0.01), axonal and nodal structural changes (p<0.001), small myelinated fiber atrophy, and degenerative changes of C‐fibers. Conclusions: GCPII inhibition has beneficial effects on hyperalgesia, nerve function, and structural degenerative changes in DPN, which are likely mediated by inhibition of ischemia‐induced glutamate release.
Differential Aggregation Of The Trembler And Trembler J Mutants Of Peripheral Myelin Protein 22
Authors: Tobler AR , Liu N , Mueller L , Shooter EM.
Published: March 2003

Mutations in the gene encoding the peripheral myelin protein 22 (PMP22), a tetraspan protein in compact peripheral myelin, are one of the causes of inherited demyelinating peripheral neuropathy. Most PMP22 mutations alter the trafficking of the PMP22 protein in Schwann cells, and this different trafficking has been proposed as the underlying mechanism of the disease. To explore this problem further, we compared the aggregation of wild‐type Pmp22 with those of the two Pmp22 mutations found in Trembler (Tr) and Trembler J (TrJ) mice. All three Pmp22s can be crosslinked readily as homodimers in transfected cells. Wild‐type Pmp22 also forms heterodimers with Tr and TrJ Pmp22, and these heterodimers traffic with their respective mutant Pmp22 homodimers. All three Pmp22s form complexes larger than dimers with Tr Pmp22 especially prone to aggregate into high molecular weight complexes. Despite the differences in aggregation of Tr and TO Pmp22, these two mutant Pmp22s sequester the same amount of wild‐type Pmp22 in heterodimers and heterooligomers. Thus, the differences in the phenotypes of Tr and TrJ mice may depend more on the ability of the mutant protein to aggregate than on the dominant‐negative effect of the mutant Pmp22 on wild‐type Pmp22 trafficking.
INFLUENCE OF EXPERIMENTAL DIABETES ON THE MICROCIRCULATION OF INJURED PERIPHERAL NERVE ‐ FUNCTIONAL AND MORPHOLOGICAL ASPECTS
Authors: Kennedy JM , Zochodne DW
Published: March 2003

Regeneration of diabetic axons has delays in onset, rate and maturation. It is possible that microangiopathy of vasa nervorum, the vascular supply of the peripheral nerve, may render an unfavorable local environment for nerve regeneration. We examined local nerve blood flow proximal and distal to sciatic nerve transection in rats with long‐term (8 month) experimental streptozotocin diabetes using laser Doppler flowmetry and microelectrode hydrogen clearance polarography. We then correlated these findings, using in vivo perfusion of an India ink preparation, by outlining the lumens of microvessels from unfixed nerve sections. There were no differences in baseline nerve blood flow between diabetic and nondiabetic uninjured nerves, and vessel number, density, and area were unaltered. After transection, there were greater rises in blood flow in proximal stumps of nondiabetic nerves than in diabetic animals associated with a higher number, density, and caliber of epineurial vessels. Hyperemia also developed in distal stumps of nondiabetic nerves but did not develop in diabetic nerves. In these stumps, diabetic rats had reduced vessel numbers and smaller mean endoneurial vessel areas. Failed or delayed upregulation of nerve blood flow after peripheral nerve injury in diabetes may create a relatively ischemic regenerative microenvironment.
COLD PRESSOR TEST DEMONSTRATES RESIDUAL SYMPATHETIC CARDIOVASCULAR ACTIVATION IN FAMILIAL DYSAUTONOMIA
Authors: Hilz MJ , Aelrod FB , Braeske B , Stemper B
Published: March 2003

In familial dysautonomia (1713), i.e. Riley‐Day‐syndrome, sympathetic cardiovascular function, as well as afferent temperature and pain mediating neurons, are significantly reduced. Thus, it was questioned if cold pressor test (CPT), which normally enhances sympathetic outflow and induces peripheral vasoconstriction by the activation of thermo‐ and nociceptive system activation, could be used to assess sympathetic function in FD. To evaluate whether CPT can be used to assess sympathetic activation in FD, we performed CPT in 15 FD patients and IS controls. After a 35‐min resting period, participants immersed their right hand and arm up to the elbow into 0‐1°C cold water while we monitored heart rate (HR), respiration, beat‐to‐beat radial artery blood pressure (BP), and laser Doppler skin blood flow (SBF) at the right index finger pulp. From these measurements, heart rate variability parameters were calculated: root mean square of successive differences (RMSSD), coefficient of variation (CV), low and high frequency (LF, HF) power spectra of the electrocardiogram (ECG). All participants perceived cold stimulation and indicated discomfort. In controls, SBF decreased and HR and BP increased rapidly upon CPT. After 60 s, SBF indicated secondary vasodilatation in six controls, BP rise attenuated and HR returned to baseline in all controls. In the patients, SBF remained unchanged, HR and BP increased significantly, but after 50‐60 s of CPT and changes were lower than in controls (p*LT0.05). RMSSD and CV decreased and LF increased significantly only in the controls. We conclude that CPT activates sympathetic HR and BP modulation despite impaired pain and temperature perception in FD patients. BP increase in the presence of almost unchanged SBF might be due to HR increase and to nociceptive arousal and emotionally induced catecholamine release as seen in emotional crises of FD patients. CPT assesses sympathetic cardiovascular responses independently from baroreflex function, which is compromised in FD.
MYELIN PROTEIN P‐0‐SPECIFIC IGM PRODUCING MONOCLONAL B CELL LINES WERE ESTABLISHED FROM POLYNEUROPATHY PATIENTS WITH MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE (MGUS)
Authors: Kvarnstrom M , Sidorova E , Nilsson J , Ekerfelt C , Vrethem M , Soderberg O , Johansson M , Rosen A , Ernerudh J.
Published: March 2003

Monoclonal expansion of B cells and plasma cells, producing antibodies against ‘self’ molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenstroms'macroglobulinaemia and B‐type of chronic lymphocytic leukaemia (B‐CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN‐MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin‐specific B cell clones from peripheral blood of patients with PN‐MGUS, by selection of cells bearing specific membrane Ig‐receptors for myelin protein P‐0, using beads coated with P‐0. P‐0‐coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein‐Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti‐P‐0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5(+) positive, although the expression declined in vitro over time. The anti‐P‐0 antibodies were of IgM‐lambda type. The antibodies belonged to the V(H)3 gene family with presence of somatic mutations. The IgM reacted with P‐0 and myelin‐associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5(+) clones included in the study as controls. The expanded clones expressed CD80 and HLA‐DR, which is compatible with properties of antigen‐presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P‐0‐specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen‐presentation by these cells followed by T cell activation.
NOTES ON THE MARINE ALGAE OF THE BERMUDAS. 3. AVRAINVILLEA SYLVEARLEAE, DISCOSPORANGIUM MESARTHROCARPUM, AND PEYSSONNELIA VALENTINII
Authors: Schneider Craig W. , Searles Richard B.
Published: March 2003

Three species of marine algae are reported from the Bermuda islands for the first time, Avrainvillea sylvearleae D. S. Littler et Littler, Discosporangium mesarthrocarpum (Menegh.) Hauck, and Peyssonnelia valentinii Yonesh. et Boudour. The presence of Avrainvillea sylvearleae in Bermuda prompted a taxonomic reinvestigation of North Carolina specimens attributed recently to that taxon, and we find them better placed in A. longicaulis f. laxa D. S. Littler et Littler. The report of the eastern Atlantic/Mediterranean Discosporangium mesarthrocarpum is the first from the western Atlantic Ocean and joins only 12 other amphi‐Atlantic species with strictly warm‐temperate zone distributions. Peyssonnelia valentinii is discovered for the first time outside of Brazil.
A NEW METHOD FOR FIXATION OF UNMINERALIZED HAPTOPHYTES FOR TEM (WHOLE MOUNT) INVESTIGATIONS
Authors: Jensen Marianne Østergaard
Published: March 2003

A new fixation method for unmineralized haptophytes is presented. Water samples are fixed with a mixture of Lugol’s solution and glutaraldehyde (final concentration 1% Lugol and 0.25% glutaraldehyde). This method preserves flagella, haptonema, and body scales very well, making light microscopical quantification at the genus level possible as well as species identification in the transmission electron microscope. The method has been tested on cultured material and natural water samples (salinities 5–34 psu).
LIFE HISTORY AND IN SITU GROWTH RATES OF ALEXANDRIUM TAYLORI (DINOPHYCEAE, PYRROPHYTA)
Authors: Garcés Esther , Delgado Maximino , Masó Mercedes , Camp Jordi
Published: March 2003

Alexandrium taylori Balech is a phototrophic marine dinoflagellate. It produced recurrent blooms during the summer months (July and August) of 1994 to 1997 in La Fosca beach (NW Mediterranean). In addition to a motile vegetative form, A. taylori had two benthic forms: temporary cysts and resting cysts. Temporary cysts were a temporally quiescent stage produced from the ecdysis of the vegetative cell in both natural populations and laboratory cultures. Temporary cysts may divide to form motile cells. Resting cysts had a thicker wall than the temporary cysts and had a red accumulation body. Gametes and planozygotes were also observed in laboratory cultures. Alexandrium taylori showed in situ diurnal vertical migration with an increase of vegetative cells in the water column in the morning through midday, with concentrations peaking in the afternoon followed by lower levels at night. Most vegetative cells lost their thecae and flagella, and with them their motility, turning into temporary cysts that settled in the early evening. The number of temporary cysts in the water column rose in the evening and at night. The temporary cysts gave rise to motile cells the following morning. Synthesis of DNA occurred in vegetative cells at night, and a preferential period of cell division occurred at sunrise. The estimated division rate in the field was 0.4–0.5 vegetative cells·day−1. Temporary cysts had twice the DNA of a G1 vegetative cell. The minimum in situ division rate of the temporary cysts was 0.14 day−1. The role of the resting and temporary cyst population in the annual recurrence and maintenance of the A. taylori bloom is discussed.
LISTENING TO PHYTOPLANKTON: MEASURING BIOMASS AND PHOTOSYNTHESIS BY PHOTOACOUSTICS
Authors: Dubinsky Zvy , Feitelson Jehuda , Mauzerall David C.
Published: March 2003

A simple technique based on photoacoustic measurements allowed us to determine the biomass, as well as the efficiency of photosynthesis, for different taxa of phytoplankton in situ. The method yields rapid, direct results compared to tedious standard measurements of 14C fixation and oxygen evolution or compared to indirect results from measurement of fluorescence. The procedure has a high potential for following the effects of environmental parameters such as irradiance, nutrient status, and pollution on phytoplankton communities and their photosynthetic activity.
LIFE ON THE EDGE: STRESS SURVIVAL ADAPTATIONS IN SOUTHERN LIMIT MACROCYSTIS PYRIFERA POPULATIONS
Authors: Ladah L.B.
Published: March 2003

In spite of subtropical temperatures often occurring at the Macrocystis pyrifera distributional southern limit in the Northern Hemisphere in Baja California, Mexico, the population has been successful at surviving and recovering from disturbance. To explore the physiological characteristics of southern limit M. pyrifera, microscopic and macroscopic phases of southern limit plants were tested for stress tolerance and compared to northern plant responses in three experiments. In spore development experiments, under constant temperature and nitrate stress treatments, spores from the southern‐limit population survived higher temperatures, had greater survivorship, and were able to complete their life cycle at higher temperatures and lower nitrate concentrations than northern population spores. During stress‐recovery treatments, where all microscopic stages were tested for recovery after stress shock exposure (temperature, nitrate and light stress), 96% of all haploid treatments showed some regrowth during recovery; haploid survival was not significantly affected by spore population origin, light, or nitrate. However, only 26% of diploid treatments showed regrowth and diploid survival was significantly affected by spore population origin; only the southern limit spores recovered after stress treatment exposure. In juvenile transplant experiments, under all conditions, juvenile plants originating from the southern‐limit had higher tissue nitrogen content, greater frond addition rates, and higher photosynthetic efficiency than northern plants, particularly at higher temperatures. The physiological differences observed for the southern limit plants versus those from the north contributes to our understanding of how the southern limit plants can survive in an environment that might otherwise be considered extreme for the species.
CLONING, SEQUENCING AND EXPRESSION OF A HISTONE‐LIKE PROTEIN FROM THE PHOTOSYNTHETIC DINOFLAGELLATE GONYAULAX POLYEDRA
Authors: Fagan F. T. , Li J.F. , Chudnovsky J. , Rizzo P.J. , Hastings J.W.
Published: March 2003

The presence of small basic DNA‐binding proteins, or histone‐like proteins (HLPs), in dinoflagellate nuclei has been well documented. Genes for HLPs have been cloned from Crypthecodinium cohnii (HCc 1 and 2) and Alexandrium fundyense (HAf ) but their predicted protein sequences show no homology to histones from other eukaryotes or to bacterial HLPs. The precise role of these HLPs in dinoflagellate nuclei is uncertain; the HLP:DNA ratio is too low to facilitate packaging of the entire (very large) genome. In order to investigate the role of these proteins we have cloned a gene for a HLP from Gonyaulax polyedra (HGp). Unlike the genes from C. cohnii, the G. polyedra gene contains no introns. It codes for a protein with a predicted molecular mass of 10,985 Da, and an isoelectric point of 10.5. We over‐expressed this protein in Escherichia coli using a construct that expresses the full‐length protein with an N‐terminal histidine tag, and purified it by Nickel‐affinity and cation‐exchange chromatography. While the predicted molecular mass of the fusion protein is 12,973 Da, similar to sizes observed for acid‐soluble G. polyedra nuclear proteins, the relative mobility on denaturing SDS‐polyacrylamide gel electrophoresis is 20,000 Da. We attribute the slower mobility in denaturing page to the basicity of the protein and predict that the relatively faster mobility exhibited by native HGp is due in part to post‐translational modification of basic residues. Such modifications have been observed in histones and play an important role in the regulation of transcription in eukaryotes, a function that HLPs may assume in dinoflagellates. This possibility is being investigated.
REACTIVE OXYGEN PRODUCTION AND DAMAGE IN INTERTIDAL FUCUS SPP. (PHAEOPHYCEAE)
Authors: Collén Jonas , Davison Ian R.
Published: March 2003

The research described in this paper was designed to test the hypothesis that the differential stress tolerance associated with the vertical zonation of intertidal seaweeds is attributable to reactive oxygen metabolism. To do so, we measured the production of, and damage caused by, reactive oxygen in three species of intertidal brown seaweeds—Fucus spiralis L., F. evanescens C. Ag., and F. distichus L.— that differ in their ability to withstand freezing, desiccation, and high light stress. Fucus spiralis is the most stress‐tolerant species and F. distichus the least. Reactive oxygen production was determined by measuring the production of H2O2 and the oxidation of dichlorohydrofluorescein diacetate to dichlorofluorescein. Damage caused by freezing, desiccation, and high‐light stress was assessed by measuring variable fluorescence (Fv/Fm) and lipid peroxidation. Production of reactive oxygen increased following freezing, desiccation, or high‐light stress. In general, the data were consistent with the hypothesis that reactive oxygen metabolism is involved in stress tolerance. The production of reactive oxygen was relatively low in unstressed seaweeds, and there was little difference between species. Fucus distichus showed the greatest increase in reactive oxygen production after desiccation and freezing stress. Fucus evanescens produced more reactive oxygen production after desiccation than F. spiralis. Although F. evanescens and F. spiralis produced similar amounts of reactive oxygen after freezing, this treatment resulted in an increase in lipid peroxidation only in F. evanescens (and F. distichus).
CELL‐WALL FORMATION DURING THE CELL CYCLE OF PORPHYRIDIUM SP. (RHODOPHYTA)
Authors: Simon‐Bercovitch Bracha , Bar‐Zvi Dudy , Arad Shoshana (Malis)
Published: March 2003

The cell wall of the red microalgae Porphyridium sp. (UTEX 637) comprises a complex amorphous polysaccharide (6–7 × 106 Da). The polysaccharide is made up of xylose, glucose, and galactose as the main sugars, as well as some minor sugars, protein, sulfate, and glucuronic acid, the latter two conferring a negative charge on the polysaccharide. In this study, we used synchronized cultures as one of the ways of unraveling the mechanism of biosynthesis of this complex polysaccharide by following cell‐wall formation during the cell cycle. Synchronization of Porphyridium sp. was achieved with an alternating light:dark regime of 12:12 h LD and dilution of the culture at the end of the cycle. Under these conditions, cell duplication occurred between the 12th and 14th hours of the cycle. The following order of building toward formation of the final polysaccharide appeared to take place: Intermediate polysaccharides with molecular masses ranging from 0.5 × 106 to 2 × 106 Da appeared in succession during hours 2–6 of the cycle, and the full‐sized polysaccharide was detected by the 8th hour. At the beginning of the cycle, xylose was the predominant sugar. Sulfur peaked at hours 2–4; glucose, galactose, and glucuronic acid at hours 8–12; and the minor sugars at hours 12–14. Upon incubation of low molecular mass polymer (0.5 × 106 Da) collected from the 4th hour with cellular crude extract from cells of the 6th hour of the cycle, two intermediates were formed (0.8 × 106 Da and 2 × 106 Da). We suggest that the 0.5 × 106 Da polymer intermediate, which is composed mainly of xylose, is the first polymer secreted into the medium, where it is further polymerized enzymatically to produce the 2 × 106 Da polymer via an intermediate 0.8 × 106 Da polymer. Later, the full‐size polysaccharide is produced.
TARGETING AND COVALENT MODIFICATION OF CELL WALL AND MEMBRANE PROTEINS HETEROLOGOUSLY EXPRESSED IN THE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE)
Authors: Fischer Harald , Robl Ingrid , Sumper Manfred , Kröger Nils
Published: March 2003

Diatoms are unicellular organisms encased by silica‐based cell walls that display species‐specific structures. Morphogenesis of diatom cell walls is believed to be controlled by a polysaccharide/protein‐matrix that remains associated with mature cell walls. Recently, a family of calcium‐binding glycoproteins, the frustulins, has been identified as major diatom cell wall component. Here we describe a transformation‐based approach to investigate intracellular targeting and function of frustulins. When ε‐frustulin from the diatom Navicula pelliculosa is expressed in Cylindrotheca fusiformis, it is correctly targeted into the cell wall. Furthermore, the unique N‐terminus of ε‐frustulin was properly modified, indicating that C. fusiformis and N. pelliculosacontain homologous frustulin‐processing proteases. In a different transformation experiment, a modified version of theChlorella kessleri hexose/H+ symporter bearing a bacterial biotinyl‐acceptor domain was expressed in C. fusiformis. The transporter became biotinylated in vivo and was functionally incorporated into the plasma membrane, allowing C. fusiformis to take up 14C‐glucose and 14C‐glucosamine. Stage‐specific radioactive labeling with this transformant revealed that secretion of frustulins is strongly enhanced during cell wall development. The data presented in this study demonstrate for the first time functional expression of a membrane protein and correct targeting of a cell wall protein heterologously expressed in a diatom cell.
NOTE ESTABLISHMENT OF AXENIC CULTURES OF ANABAENA FLOS‐AQUAE AND APHANOTHECE NIDULANS (CYANOBACTERIA) BY LYSOZYME TREATMENT
Authors: Kim Jung‐Seok , Park Yong‐Ha , Yoon Byung‐Dae , Oh Hee‐Mock
Published: March 2003

A new method using lysozyme for the production of axenic cultures of Anabaena flos‐aquae De Brebisson and Aphanothece nidulans P. Richter was developed. Cyanobacterial growth was not inhibited at concentrations up to 1.2 g·L−1 of lysozyme, whereas the growth of heterotrophic bacteria was suppressed. At concentrations up to 0.8 g·L−1 of lysozyme, ampicillin caused a reduction of heterotrophic bacteria. The axenic cultures of these strains were acquired through a simple treatment using 1.0 g·L−1 of lysozyme without ampicillin. These cyanobacteria resisted digestion by lysozyme at our experimental concentrations, whereas bacteria were digested selectively. This method of purification seems to be especially useful with cyanobacterial species that are sensitive to antibiotics or other germicidal agents.
HEAVY‐METAL INDUCED CHANGES IN NONPROTEINACEOUS THIOL LEVELS AND HEAVY‐METAL BINDING PEPTIDE IN TETRASELMIS TETRATHELE (PRASINOPHYCEAE)
Authors: Satoh Masaya , Karaki Emi , Kakehashi Mamiko , Okazaki Eisuke , Gotoh Toshio , Oyama Yasuo
Published: March 2003

The effects of mercury and cadmium on the intracellular level of nonproteinaceous thiols in a unicellular green alga Tetraselmis tetrathele (West) Butcher (Prasinophyceae) were investigated by using a fluorescent dye, 5‐chloromethylfluorescein (5CMF), as a probe for nonproteinaceous thiols. The 5CMF fluorescence was observed in cytoplasm, and the intensity of the fluorescence was decreased by exposure of the cells to HgCl2. Analysis of the fluorescent intensity of 5CMF by flow cytometry made it possible to distinguish cells in three states during the dying process caused by HgCl2: a normal state, a thiol‐depleted state, and a dead state. Depletion of nonproteinaceous thiols began within 30 min, and they were completely depleted at 2 h. Most cells died after 24 h of exposure to more than 3.0 μM HgCl2, whereas exposure up to 1.0 mM CdCl2 did not cause depletion of nonproteinaceous thiols or cell death within 48 h.  HPLC analyses revealed that glutathione was a major nonprotein thiol in T. tetrathele and that it was oxidized by exposing the cells to HgCl2. Phytochelatins, which play a great role in the tolerance to heavy metals of higher plants and many algae, could not be found in T. tetrathele. However, a tripeptide, Arg‐Arg‐Glu, was found to be abundant, and it showed ability to bind Hg2+, suggesting that it functions to scavenge heavy metals as well as thiol molecules.
LOCATION AND EXPRESSION OF FRUSTULINS IN THE PENNATE DIATOMS CYLINDROTHECA FUSIFORMIS, NAVICULA PELLICULOSA, AND NAVICULA SALINARUM (BACILLARIOPHYCEAE)
Authors: Poll Willem H. van de , Vrieling Engel G. , Gieskes Winfried W. C.
Published: March 2003

A detailed immunocytochemical and biochemical study of the location and expression of frustulins, a family of proteins associated with the frustules of diatoms, has been performed for Cylindrotheca fusiformis Reimann et Lewin, Navicula pelliculosa (Brébisson et Kützing) Hilse, and Navicula salinarum (Grunow) Husted. Immunocytochemistry revealed that frustulins, which share homologous epitopes but are different in size, were predominantly located in the organic casing. Based on timed immunolocalization experiments and Western blotting analysis of cell extracts obtained sequentially after repleting silicate to Si‐synchronized cells, the continuous presence of the frustulins in the mature and parental organic casing of the examined species was observed. The frustulins of N. pelliculosa appeared as proteins similar to those of C. fusiformis, sharing identical epitopes. The extractions, however, yielded a markedly lower abundance of frustulins in N. pelliculosa. Peak concentrations of extracted frustulins appeared to be expressed just ahead of the silicification process in C. fusiformis, whereas the level of expression in N. pelliculosa increased along with maturation of the new valves. For N. salinarum, the presence of the frustulins could not be confirmed properly by Western blotting, most probably because of the small sample volumes, inefficient extraction, and a lower amount of homologous frustulins in the casing of this species. It is concluded that the frustulins of these species are not associated with the silicalemma of the newly formed silica deposition vesicles and therefore do not seem to be involved in the silicification process itself. Overall, the results imply a structural role of the frustulins in the casing of diatoms rather than a regulation of the silicification process.
GEOTAXIS/PHOTOTAXIS AND BIOCHEMICAL PATTERNS IN HETEROCAPSA (=CACHONINA) ILLDEFINA (DINOPHYCEAE) DURING DIEL VERTICAL MIGRATIONS
Authors: Kamykowski Daniel , Milligan Edward J. , Reed Robert E. , Liu Wenchen
Published: March 2003

Two separate experiments with Heterocapsa (=Cachonina) illdefina Herman et Sweeney, one with and the other without water volume replacement, were performed in a 250‐L laboratory mesocosm (45‐cm diameter × 150‐cm height) to examine how diel vertical migration (DVM) relates to taxis sign and strength and to cellular biochemical state. Although only the cell population grown with water volume replacement maintained a division per day over the course of the experiment, periodic measurements during both experiments demonstrated that cells aggregating at the surface during the light period generally were deficient in all measured biochemical constituents compared to cells obtained from a midcolumn depth. More specifically, H. illdefina cells that aggregated at the surface during the light period in both experiments exhibited weakened positive geotaxis but strengthened positive phototaxis and were very deficient in lipid and free amino acid compared to midcolumn cells. Cells sampled at midcolumn during the light period exhibited similar but weaker taxes changes compared to surface samples, and geotaxis strength was inversely correlated with cell diameter, cellular DNA and protein content, and RNA/DNA ratio. In comparison, published data on Gymnodinium breve Davis, a harmful algal bloom species, showed that cells aggregating at the surface during the light period generally exhibited weakened negative geotaxis and strengthened positive phototaxis and were very deficient in lipid and chl a compared to midcolumn cells. Although the persistent tendency toward negative geotaxis was weaker in midcolumn subpopulations throughout the day, its strength was inversely correlated with cell diameter and cellular lipid content. The combined results for both species support a revised conceptual model of optimized DVM in autotrophic marine dinoflagellates incorporating generalized expressions of taxis and biochemical state of individual cells.
CONSPECIFICITY AND INDO‐PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS
Authors: Baillie Brett K. , Belda‐Baillie Carmen A. , Maruyama Tadashi
Published: March 2003

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.
ENDOMEMBRANE STRUCTURE AND THE CHLOROPLAST PROTEIN TARGETING PATHWAY IN HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE, CHROMISTA)
Authors: Ishida Ken‐ichiro , Cavalier‐Smith Thomas , Green Beverley R.
Published: March 2003

Chloroplasts in heterokont algae are surrounded by four membranes and probably originated from a red algal endosymbiont that was engulfed and retained by eukaryotic host. Understanding how nuclear‐encoded chloroplast proteins are translocated from the cytoplasm into the chloroplast across these membranes could give us some insights about how the endosymbiont was integrated into the host cell in the process of secondary symbiogenesis. In multiplastid heterokont algae such as raphidophytes, it has been unclear if the outermost of the four membranes surrounding the chloroplast (the chloroplast endoplasmic reticulum [CER] membrane) is continuous with the nuclear envelope and rough endoplasmic reticulum (ER). Here, we report detailed ultrastructural observations of the raphidophyte Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara that show that the CER membranes were continuous with ER membranes that had attached ribosomes, implying that the chloroplast with three envelope membranes is located within the ER lumen, that is, topologically the same structure as that of monoplastid heterokont algae. However, the CER membrane of H. akashiwo had very few, if any, ribosomes attached, unlike the CER membranes in other heterokont algae. To verify that proteins are first targeted to the ER, we assayed protein import into canine microsomes using a precursor for a nuclear‐encoded chloroplast protein, the fucoxanthin‐chlorophyll a/c protein of H. akashiwo. This demonstrated that the precursor has a functional signal sequence for ER targeting and is cotranslationally translocated into the ER, where a signal sequence of about 17 amino acids is removed. Based on these data, we hypothesize that in H. akashiwo, nuclear‐encoded chloroplast protein precursors that have been cotranslationally transported into the ER lumen are sorted in the ER and transported to the chloroplasts through the ER lumen.
ACTIN GENE DUPLICATION AND THE EVOLUTION OF MORPHOLOGICAL COMPLEXITY IN LAND PLANTS
Authors: Bhattacharya Debashish , Aubry John , Twait Erik C. , Jurk Stefanie
Published: March 2003

Actin is a highly conserved cytoskeletal protein that is a key component of cells. Genes encoding actin occur in single copies in most green algae, in 2–3 copies in bryophytes, and in increasingly more complex gene families in ferns and seed plants. We use the well‐resolved phylogenetic frameworks of the Streptophyta as a guide to reconstruct the patterns of actin gene duplication in early diverging land plants. Our working hypothesis is that the origin of novel tissues in the bryophytes (e.g. multicellular sporophyte) may be reflected in the functional diversification of duplicate actin genes in these taxa. Actin is used as a model cytoskeletal protein with the assumption that its evolutionary history represents those of other cytoskeletal elements and the coevolved binding proteins. Here we provide a phylogenetic perspective on the origin of green algal and land plant actin genes and use this information to speculate on the role of plant actin in early plant evolution.
Responses of crustose corallines to epiphyte and canopy cover
Authors: De O. Figueiredo Marcia A. , Kain ( Jones) Joanna M , Norton Trevor A.
Published: March 2003

Although a dense cover of epiphytes is generally considered to be harmful for some coralline algae (Corallinaceae, Rhodophyta), crustose corallines in the littoral zone seem to be preserved from bleaching when covered by canopy plants and epiphytes during summer. This study aimed to test the responses of coralline crusts to epiphytes and canopy algae and their interaction with grazing limpets. Growth rates and color changes were followed in two crust species in areas with or without canopy algae in the Isle of Man, British Isles. Limpets were excluded, to allow epiphytes to grow upon crusts. Responses were measured both on pieces of crusts upon acrylic plates and on crusts growing naturally on the shore. Fucus canopy and epiphytic Enteromorpha significantly influenced the crusts' growth, depending on season. Epiphytes reduced the light levels beneath by up to 78%, more than the canopy algae (62%). Crusts exposed outside the canopy bleached in summer, but gradually restored their color once they were covered by epiphytes. The fast‐growing Phymatolithon lenormandii (Aresch.) Adey recovered its coloration more quickly than the slow‐growing P. purpureum (P. et H. Crouan) Woelkerling et Irvine. However, neither crust species could restore its color when epiphytes were reduced by grazing limpets, Patella vulgata L. Bleaching did not kill the crusts, but seemed to interfere with crusts' growth. Restoration of pigmentation was quantified for the first time on bleached coralline crusts. Epiphyte and canopy algae were experimentally shown to be beneficial, probably by providing shade and also protecting crusts from desiccation.
Carbohydrate regulation of attachment, encystment, and appressorium formation by Pythium porphyrae (Oomycota) zoospores on Porphyra yezoensis (Rhodophyta)
Authors: Uppalapati Srinivasa Rao , Fujita Yuji
Published: March 2003

Pythium porphyrae Takahashi et Sasaki, a facultative parasite of Porphyra spp., is the common microbial agent responsible for red rot disease of this red alga in Japan. Host infection by this species and other plant parasitic members of the Pythiaceae is initiated by motile biflagellate zoospores. Factors regulating host specificity and the initial steps involved in the infection process, consisting of attachment, encystment and appressorium formation, are not known. Zoospore encystment and appressorium formation of P. porphyrae were monitored by staining of the fungal cell walls using calcofluor. The zoospores infected only Porphyra spp. and Bangia atropurpurea (Roth) C. Agardh thalli, although they attached to, and encysted on, many other members of the Rhodophyceae (Stylonema alsidii[Zanardini] Drew, Gelidium elegans Kützing, Pterocladiella capillacea[Gmelin] Santelices et Hommersand, Carpopeltis affinis[Harvey] Okamura, Gloiosiphonia capillaris[Hudson] Carmichael in Berkeley, Grateloupia turuturu Yamada, Callophyllis adhaerens Yamada, Gracilaria spp., Lomentaria hakodatensis Yendo, Rhodymenia intricata[Okamura] Okamura, Griffithsia subcylindrica Okamura, Wrangelia tanegana Harvey, and Polysiphonia morrowii Harvey). No attachment or encystment was observed on the red alga Kappaphycus striatum (Schmitz) Doty ex Silva in Silva et al., the brown algae Undaria pinnatifida (Harvey) Suringar, Scytosiphon sp., and Sargassum thunbergii (Mertens ex Roth) Kuntze as well as members of the Ulvaceae (green algae). Sequential extraction of carbohydrates from Porphyra yezoensis Ueda thalli and the addition of diverse monosaccharides, polysaccharides, and amino acids to zoospore suspensions indicated that encystment and appressorium formation were induced only by sulfated galactans (porphyran, commercial agar, agarose, and carrageenans). Zoospore attachment and encystment on thalli of P. yezoensis was abolished by periodate oxidation of the thallus surface and was reduced by 80–90% after enzymatic removal of sulfated galactan (porphyran). It appears that the interaction of zoospore surface receptors with sulfated galactan (porphyran) determinants on the thallus surface induced specific attachment and encystment on Porphyra spp. thalli. Zoospores encysted, germinated, and formed appressoria on sulfated galactan films and in suspensions of this carbohydrate. Attachment and encystment were induced on commercial agar and agarose films, but appressoria were not induced on agarose films. Supplementation of agarose media with both cold and hot water fractions and with porphyran from P. yezoensis–induced appressoria implicated sulfated galactans (porphyran) in appressorium formation.
VERTICAL MIGRATION BY RHIZOSOLENIA SPP. (BACILLARIOPHYCEAE): IMPLICATIONS FOR FE ACQUISITION
Authors: McKay R. Michael L. , Villareal Tracy A. , La Roche Julie
Published: March 2003

Available data support a mechanism of buoyancy‐mediated vertical migration by large‐sized diatoms of Rhizosolenia spp. as a means to access “new” nitrogen from deep waters. To assess whether phytoplankton simultaneously satisfy their Fe requirements by this mechanism, field samples collected during summer 1996 at stations located along a transect through the central North Pacific gyre were assayed for the presence of flavodoxin and ferredoxin via Western blot analysis. All samples, regardless of their buoyancy status and the station from which they were collected, had accumulated flavodoxin but not ferredoxin. To understand better the significance of the field results, cultures of Rhizosolenia formosa H. Peragallo were grown in the laboratory with varying levels of total Fe (200 nM–10,000 nM). Fe had little effect on the physiological and photochemical parameters measured for each treatment. Growth rates did not exceed 0.17 d−1 and values of Fv/Fm ranged from 0.48 to 0.62. In addition, R. formosa accumulated only flavodoxin at each level of Fe addition. From these results, it appears that for some rhizosolenids, flavodoxin is constitutively expressed. The underlying basis for the constitutive nature of this flavodoxin is unclear at present, although it is likely that it is ultimately related to chronic Fe deficit incurred in natural waters.
HETEROCYST DEVELOPMENT AND LOCALIZATION OF CYANOPHYCIN IN N2‐FIXING CULTURES OF ANABAENA SP. PCC 7120 (CYANOBACTERIA)
Authors: Sherman Debra M. , Tucker Don , Sherman Louis A.
Published: March 2003

The process of N2 fixation in the filamentous cyanobacterium Anabaena sp. PCC 7120 is known to occur in terminally differentiated cells called heterocysts. This study is concerned with a morphological and immunocytochemical analysis of the developing heterocysts. The heterocysts continue a developmental process after synthesis of the specialized cell wall and the formation of the proheterocyst. The initial stages were described by Wilcox et al. (1973) and designated stages 1 through 7, with stages 5–7 associated with the maturing heterocyst. We now designate a stage 8 as the postmaturation stage, based on physiological and ultrastructural evidence. Immunocytochemistry to detect the nitrogenase protein NifH and the nonribosomally synthesized polypeptide cyanophycin demonstrated a complementary accumulation of these polypeptides. Accumulation of the nitrogenase protein was greatest at stages 5 and 6 and then declined precipitously. Cyanophycin was more prevalent after late stage 6 and was primarily associated with the polar nodule (polar plug) and the neck connecting the heterocyst with the adjoining vegetative cell. We suggest that the cyanophycin‐containing polar plug is a key intermediate in the storage of fixed nitrogen in the heterocyst, a result consistent with the suggestion first made by Carr (1988) that cyanophycin exists as a dynamic reservoir of fixed nitrogen within the heterocysts.
TAXONOMY OF THE NEW ZEALAND‐ENDEMIC GENUSPLEUROSTICHIDIUM(RHODOMELACEAE, RHODOPHYTA)
Authors: Phillips Louise E.
Published: March 2003

A morphological, anatomical, and molecular study of the tribe Pleurostichidieae (Rhodomelaceae, Ceramiales) is presented. New collections of its only member,Pleurostichidium falkenbergiiHeydrich, have enabled a thorough re‐assessment of this species from a classical‐morphological standpoint and have allowed the first photographs to be made of critical features of this little‐known obligate epiphyte of the brown algaXiphophora chondrophylla(Turner) MontagneexHarvey. The relationship of the tribe to other members of the Rhodomelaceae is considered based on analysis of 18S rDNA sequences fromP. falkenbergii, 14 other rhodomelaceous species, and six outgroup taxa.Pleurostichidium falkenbergiiis shown to be most closely related to the tribe Polysiphonieae and only distantly related to the Amansieae, with which it was previously associated.