The Evolution of HD2 Proteins in Green Plants.

In eukaryotes, protein deacetylation is carried out by two well-conserved histone deacetylase (HDAC) families: RPD3/HDA1 and SIR2. Intriguingly, model plants such as Arabidopsis express an additional plant-specific HDAC family, termed type-2 HDACs (HD2s). Transcriptomic analyses from more than 1300 green plants generated by the 1000 plants (1KP) consortium showed that HD2s appeared early in green plant evolution, the first members being detected in several streptophyte green alga. The HD2 family has expanded via several rounds of successive duplication; members are expressed in all major green plant clades. Interestingly, angiosperm species express new HD2 genes devoid of a zinc-finger domain, one of the main structural features of HD2s. These variants may have been associated with the origin and/or the biology of the ovule/seed.

Frequent proviral integration of the human betaretrovirus in biliary epithelium of patients with autoimmune and idiopathic liver disease.

BACKGROUND: A human betaretrovirus (HBRV) has been linked with primary biliary cirrhosis (PBC) following the detection of viral particles in biliary epithelium by electron microscopy and cloning of the betaretrovirus genome from biliary epithelium and peri-hepatic lymph nodes. Evidence for viral infection was found in the majority of PBC patients' peri-hepatic lymph node samples. However, less than a third of the liver samples had detectable HBRV, whereas others were unable to detect betaretrovirus infection or noted the presence of virus in the liver of patients with other diagnoses. AIMS: To address the hypothesis that the betaretrovirus may be below the limits of detection in the liver, biliary epithelial cells (BEC) were investigated for the evidence of infection. METHODS: Ligation-mediated PCR and next generation sequencing were used to detect proviral integrations in liver, lymph nodes and BEC isolated from liver transplant recipients. Hybridisation-based assays were used to detect betaretroviral RNA in BEC. RESULTS: Unique HBRV integrations and betaretrovirus RNA were detected in the majority of biliary epithelia derived from patients with PBC, autoimmune hepatitis and cryptogenic liver disease but rarely in other liver transplant recipients with primary sclerosing cholangitis and other hepatic disorders. HBRV integrations were commonly found in PBC patients' lymph nodes but rarely in whole liver samples. CONCLUSIONS: Human betaretrovirus infection is frequently observed at the site of disease in patients with primary biliary cirrhosis and also in biliary epithelium of patients with autoimmune hepatitis and cryptogenic liver disease.

Amazon River enhances diazotrophy and carbon sequestration in the tropical North Atlantic Ocean.

The fresh water discharged by large rivers such as the Amazon is transported hundreds to thousands of kilometers away from the coast by surface plumes. The nutrients delivered by these river plumes contribute to enhanced primary production in the ocean, and the sinking flux of this new production results in carbon sequestration. Here, we report that the Amazon River plume supports N(2) fixation far from the mouth and provides important pathways for sequestration of atmospheric CO(2) in the western tropical North Atlantic (WTNA). We calculate that the sinking of carbon fixed by diazotrophs in the plume sequesters 1.7 Tmol of C annually, in addition to the sequestration of 0.6 Tmol of C yr(-1) of the new production supported by NO(3) delivered by the river. These processes revise our current understanding that the tropical North Atlantic is a source of 2.5 Tmol of C to the atmosphere [Mikaloff-Fletcher SE, et al. (2007) Inverse estimates of the oceanic sources and sinks of natural CO(2) and the implied oceanic carbon transport. Global Biogeochem Cycles 21, doi:10.1029/2006GB002751]. The enhancement of N(2) fixation and consequent C sequestration by tropical rivers appears to be a global phenomenon that is likely to be influenced by anthropogenic activity and climate change.

Non-Gaussian statistics of the vibrational fluctuations of myoglobin.

Experiments on the dynamics of vibrational fluctuations in myoglobin revealed an interesting behavioral cross-over occurring in the range 180-200 K. In this temperature range the mean square displacement of atomic positions versus temperature sharply increases its slope, indicating the dissociation of CO from the heme group. In this paper we develop a theoretical model that provides a framework for the quantitative description of this phenomenon. The basis of our calculations is an assumption of an effective potential with multiple local minima. In particular, we consider a quartic potential in place of the simple quadratic. We then use non-Gaussian statistics to obtain a relationship between the mean square displacement and model parameters. We compare our model to published experimental data and show that it can describe the data set using physically meaningful parameters which are fitted to the experimental data. In the process we verify the Gaussian approximation's applicability only to the low-temperature régime. In the high-temperature limit, however, deviations from the Gaussian approximation are due to the double-well nature of our effective potential. We find that the published datasets showing the thermal transition display the qualitative trends predicted by appropriate algebraic approximations to our predicted myoglobin behavior.

Buoyancy regulation and the potential for vertical migration in the oceanic cyanobacterium trichodesmium.

Diel protein and carbohydrate content in Trichodesmium thiebautii was measured to evaluate the relationship to buoyancy status. Carbohydrate:protein ratio was the best predictor of buoyancy and fit a cosine curve with increasing values during the day and decreasing values at night in cycles that paralleled observed diel buoyancy patterns. This ratio also increased in short-term experiments as a function of light and increased in parallel with decreasing positive buoyancy. We used changes in this ratio to estimate the potential for vertical migration. Whereas limited vertical excursions in the upper 70 m are possible, deeper migrations appear unlikely unless respiration rates decrease significantly. N:P ratios in sinking and floating colonies were used to test for the P acquisition at depth (vertical migration). We noted that pooled N:P ratios were not significantly different between sinking and ascending colonies (N:P = 65.6 and 66.3, respectively) collected along the northern Australian coast, much like published results from north of Hawaii. Highly significant differences (p <0.0001) were observed in the western Gulf of Mexico between sinking and ascending colonies (N:P = 87.0 and 43.5, respectively) and provide the best direct evidence to date of vertical migration for P acquisition. Our physiological data on compositional changes during buoyancy reversals suggest a complex relationship between light and nutrients. It appears likely that light and P metabolism interact to regulate the vertical extent of migrations, with deep vertical migration regulated by P metabolism superimposed on a mixed-layer light-driven migration. The variability in N:P ratios suggests that care should be taken in assuming buoyancy reversals always result in P acquisition in this oceanic cyanobacterium.

Phosphorus limitation of nitrogen fixation by Trichodesmium in the central Atlantic Ocean.

Marine fixation of atmospheric nitrogen is believed to be an important source of biologically useful nitrogen to ocean surface waters, stimulating productivity of phytoplankton and so influencing the global carbon cycle. The majority of nitrogen fixation in tropical waters is carried out by the marine cyanobacterium Trichodesmium, which supplies more than half of the new nitrogen used for primary production. Although the factors controlling marine nitrogen fixation remain poorly understood, it has been thought that nitrogen fixation is limited by iron availability in the ocean. This was inferred from the high iron requirement estimated for growth of nitrogen fixing organisms and the higher apparent densities of Trichodesmium where aeolian iron inputs are plentiful. Here we report that nitrogen fixation rates in the central Atlantic appear to be independent of both dissolved iron levels in sea water and iron content in Trichodesmium colonies. Nitrogen fixation was, instead, highly correlated to the phosphorus content of Trichodesmium and was enhanced at higher irradiance. Furthermore, our calculations suggest that the structural iron requirement for the growth of nitrogen-fixing organisms is much lower than previously calculated. Although iron deficiency could still potentially limit growth of nitrogen-fixing organisms in regions of low iron availability-for example, in the subtropical North Pacific Ocean-our observations suggest that marine nitrogen fixation is not solely regulated by iron supply.

Separation of proteins by sodium dodecylsulfate capillary electrophoresis in hydroxypropylcellulose sieving matrix with laser-induced fluorescence detection.

Sodium dodecyl sulfate capillary electrophoresis by using hydroxypropylcellulose as the sieving matrix was developed for separation of proteins. 3-(2-furoyl)quinoline-2-carboxaldehyde, a fluorogenic dye, was used as the pre-column reagent to label proteins, which allows the use of laser-induced fluorescence to improve the detection sensitivity. Five standard proteins within the molecular mass range of 14,000-97,000 were used to test this method and a calibration curve was obtained between the molecular mass of these proteins and their peak migration times. This method was also applied to the separation of proteins from HT29 human colon adenocarcinoma cell extracts, and, typically, nearly 30 protein components could be resolved in a 20-min separation. Similar separation patterns were observed for the cell extract proteins when three running buffer systems were employed, indicating that buffer composition did not have much influence on the separation based on HPC sieving.

Bacterial activity in South Pole snow.

Large populations (200 to 5,000 cells ml(-1) in snowmelt) of bacteria were present in surface snow and firn from the south pole sampled in January 1999 and 2000. DNA isolated from this snow yielded ribosomal DNA sequences similar to those of several psychrophilic bacteria and a bacterium which aligns closely with members of the genus Deinococcus, an ionizing-radiation- and desiccation-resistant genus. We also obtained evidence of low rates of bacterial DNA and protein synthesis which indicates that the organisms were metabolizing at ambient subzero temperatures (-12 to -17 degrees C).

New perspectives on nitrogen-fixing microorganisms in tropical and subtropical oceans.

New molecular and microscopic evidence indicates that the open ocean harbors a diverse range of novel free-living and symbiotic nitrogen-fixing microorganisms. Although the extent to which these microorganisms fix nitrogen is currently unclear, ongoing research indicates that they might make a substantial contribution to the open ocean nitrogen budget.

Molecular cloning and antiserum development of cyclin box in the brown tide alga Aureococcus anophagefferens.

Cyclins can be useful cell cycle markers for growth rate studies on harmful algal blooms. In this study, a gene fragment corresponding to cyclin box was cloned for the brown tide alga Aureococcus anophagefferens. This algal gene fragment, designated as Btcycl1, was most similar to cyclin B. Oligopeptides based on the deduced amino acid sequence were synthesized and used to raise an antiserum that reacted on Western blots with a protein of about 63 kDa, the same size as cyclin B in other organisms. The cyclin B-like protein recognized by this antiserum, and the messenger RNA amplified using the primers, were more abundant in exponential cultures and decreased markedly in stationary cultures. This protein also appeared to be cell cycle dependent. Immunofluorescence labeling showed that this antiserum specifically stained a protein in Aureococcus cells and had no cross-reaction with bacteria that were present in the algal culture. The Btcycl1 sequence and the antiserum will provide a useful tool for studies on regulation of in situ growth rate for this brown tide alga.

A PSTTLRE-form of cdc2-like gene in the marine microalga Dunaliella tertiolecta.

To understand the genetic control of algal cell division cycle that pertains to phytoplankton bloom dynamics in the sea, we cloned and analyzed a gene coding for a cyclin-dependent kinase (CDK) for the chlorophyte Dunaliella tertiolecta. The cDNA cloned, 1061 bp long, contained an open reading frame of 314 amino acids. FASTA and GAP analyses showed that this sequence was most homologous to cdc2 out of all known cdks, with an identity of 54-68% and a similarity of 65-76% to cdc2 in higher plants, animals, and yeast. Several signature domains of cdc2 were identified from this sequence, although the PSTAIRE and GDSEID motifs were replaced with PSTTLRE and GDCELQ, respectively. Southern blot hybridization demonstrated that this gene occurred as a single copy in this species, and quantitative RT-PCR showed that the transcription of this gene was constitutive. The present results suggest that the universal cdc2 is conserved in the lower eukaryote with unique structural characteristics.

Host specificity in the Richelia-diatom symbiosis revealed by hetR gene sequence analysis.

The filamentous heterocyst-forming cyanobacterium Richelia intracellularis forms associations with diatoms and is very abundant in tropical and subtropical seas. The genus Richelia contains only one species, R. intracellularis Schmidt, although it forms associations with several diatom genera and has considerable variation in size and morphology. The genetic diversity, and possible host specificity, within the genus Richelia is unknown. Using primers against hetR, a gene unique for filamentous cyanobacteria, specific polymerase chain reaction (PCR) products were obtained from natural populations of R. intracellularis filaments associated with three diatom genera. Phylogenetic analyses of these sequences showed that they were all in the same clade. This clade contained only the R. intracellularis sequences. The genetic affiliation of hetR sequences of R. intracellularis to those of other heterocystous cyanobacteria strongly suggests that it was not closely related to endosymbiotic Nostoc spp. hetR sequences. Sequences from R. intracellularis-Hemiaulus membranaceus sampled in the Atlantic and Pacific Oceans were almost identical, demonstrating that the genetic relatedness was not dependent on geographical location. All sequences displayed a deep divergence between symbionts from different genera and a high degree of host specificity.

Identification and preliminary characterization of PCNA gene in the marine phytoplankton Dunaliella tertiolecta and Isochrysis galbana.

The gene coding for proliferating cell nuclear antigen (PCNA) was identified in Dunaliella tertiolecta (Chlorophyceae) and Isochrysis galbana (Prymnesiophyceae). Southern blot hybridization using a PstI fragment of rat PCNA gene (pCR-1) as a probe showed that there is apparently a single copy of this gene per haploid genome in both species. On the Northern blot pCR-1 probed a single messenger RNA for each species of a molecular size close to rat PCNA mRNA (1.1 kilobases [kb]). Polymerase chain reaction (PCR) with a set of degenerated primers produced a fragment of about 610 base pairs [bp] from genomic DNA of both species; the PCR products appeared close in size to the amplified from rat PCNA and hybridized to the pCR-1 probe. Further analysis with reverse transcription-PCR (RT-PCR), cloning, and sequencing revealed a complementary DNA of a similar size (616 and 576 bp) that possesses an open reading frame encoding 205 and 192 amino acids, respectively, for Dun (D. tertiolecta) and Iso (I. galbana). Surprisingly, the polypeptides deduced from the two cDNA shared no higher homology to each other (71%) than to animals such as Xenopus (Dun 72%; Iso 73%), rat (Dun 73%; Iso 74%), and human (Dun 73%; Iso 74%), and to higher plants such as soybean (Dun 78%; Iso 72%), Zea mays (Dun 77%; Iso 73%), and rice (Dunn 77%; Iso 72%), although D. tertiolecta has a higher homology (77%) to the Prasinophyceae alga Tetraselmis chiu than does I. galbana (71%). The homology to PCNA in budding and fision yeasts (63% and 53%, respectively) is also lower than to animals and higher plants. It is thus suggested that with regard to PCNA genes, the three algae are as different from each other as they are from higher plants and animals. In a partially synchronized exponential culture of D. tertiolecta grown with a photocycle of 12 h light and 12 h dark, the abundance of the transcript appeared to be low at hours 3 and 9 (hour 0 = the onset of light period), and increased about 2- to 3-fold at hours 15 and 21 (i.e., during the dark period). Western blotting and immunofluorescence analysis on concurrent diel samples showed an over 2-fold increase in PCNA protein abundance (in proportion to total cellular protein) and the percentage of cells labeled by PCNA antibody. A similar trend was found for I. galbana grown under the same conditions. The results suggest that the gene transcription was in pace with PCNA synthesis, which was lower in the light period when G1 phase was dominant and higher in the dark period when S (and probably G2 and early M) phase was dominant, and that the expression of this gene may be regulated at the transcriptional level in these two algae.

Amino Acid Cycling in Colonies of the Planktonic Marine Cyanobacterium Trichodesmium thiebautii.

We examined diel trends in internal pools and net efflux of free amino acids in colonies of the nonheterocystous, diazotrophic cyanobacterium Trichodesmium thiebautii, freshly collected from waters of the Caribbean and the Bahamas. The kinetics of glutamate uptake by whole colonies were also examined. While intracolonial pools of most free amino acids were relatively constant through the day, pools of glutamate and glutamine varied over the diel cycle, with maxima during the early afternoon. This paralleled the daily cycle of nitrogenase activity. We also observed a large net release of these two amino acids from intact colonies. Glutamate release was typically 100 pmol of N colony h. This is about one-fourth the concurrent rate of N(2) fixation during the day. However, while nitrogenase activity only occurs during the day, net release of glutamate and glutamine persisted into the night and may therefore account for a greater loss of recently fixed N on a daily basis. This release may be an important route of new N input into tropical, oligotrophic waters. Whole colonies also displayed saturation kinetics with respect to glutamate uptake. The K(s) for whole colonies varied from 1.6 to 3.2 muM, or about 100-fold greater than typical ambient concentrations. Thus, uptake systems appear to be adapted to the higher concentrations of glutamate found within the intracellular spaces of the colonies. This suggests that glutamate may be a vehicle for N exchange among trichomes in the colony.

Cytochrome Oxidase: Subcellular Distribution and Relationship to Nitrogenase Expression in the Nonheterocystous Marine Cyanobacterium Trichodesmium thiebautii.

Immunochemical labeling was used to study the subcellular distribution of cytochrome oxidase, a respiratory protein, in Trichodesmium thiebautii. The protein was found associated with both cytoplasmic and thylakoid membranes. About a sixfold variation in the protein content (gold particle count) was found among Trichodesmium cells within a single colony. Double labeling was performed with cytochrome oxidase and nitrogenase antisera. Regression analysis of gold particle counts per unit of cell area of cytochrome oxidase and nitrogenase showed a positive correlation (r = 0.911); cells with higher nitrogenase levels also had higher levels of cytochrome oxidase. The parallel expression of two proteins suggests that respiratory oxygen uptake may be involved in nitrogenase protection (respiratory protection) in Trichodesmium spp.

Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp.

We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation.

Ultrastructural and chemical assessment of poly-beta-hydroxybutyric acid in the marine cyanobacterium Trichodesmium thiebautii.

We report here on the occurrence and quantities of poly-beta-hydroxybutyric acid (PHB) in natural populations of the marine cyanobacterium Trichodesmium thiebautii. A diurnal variation in the shape and size of PHB granules and in PHB content was observed. The highest PHB levels (2.3 +/- 0.8 mg g-1 dry wt) were recorded in the early morning and the values decreased thereafter with a minimum at night (1.6 +/- 0.9 mg g-1 dry wt). Our data suggest that PHB is a prominent cell constituent in T. thiebautii and that its synthesis takes place in the early morning whereas it is utilized during the rest of the day.

Major role of the cyanobacterium trichodesmium in nutrient cycling in the north atlantic ocean.

The diazotrophic cyanobacterium Trichodesmium is a large (about 0.5 by 3 millimeters) phytoplankter that is common in tropical open-ocean waters. Measurements of abundance, plus a review of earlier observations, indicate that it, rather than the picophytoplankton, is the most important primary producer (about 165 milligrams of carbon per square meter per day) in the tropical North Atlantic Ocean. Furthermore, nitrogen fixation by Trichodesmium introduces the largest fraction of new nitrogen to the euphotic zone, approximately 30 milligrams of nitrogen per square meter per day, a value exceeding the estimated flux of nitrate across the thermocline. Inclusion of this organism, plus the abundant diazotrophic endosymbiont Richelia intracellularis that is present in some large diatoms, in biogeochemical studies of carbon and nitrogen may help explain the disparity between various methods of measuring productivity in the oligotrophic ocean. Carbon and nitrogen fixation by these large phytoplankters also introduces a new paradigm in the biogeochemistry of these elements in the sea.

Basis for Diel Variation in Nitrogenase Activity in the Marine Planktonic Cyanobacterium Trichodesmium thiebautii.

Natural populations of the nonheterocystous marine cyanobacterium Trichodesmium thiebautii exhibit a diel periodicity in nitrogenase activity (NA). NA "turns on" near dawn and "turns off" near dusk, independent of photic conditions. Chloramphenicol (CAP) and ammonium prevented turn on of NA in T. thiebautii when added to samples collected before dawn but were progressively less effective in inhibiting NA in samples collected later in the morning. In samples collected after turn on, activities declined with time with both CAP and ammonium treatments, with ammonium having a stronger effect. In contrast, CAP added to samples collected in late afternoon prolonged NA, compared with controls, which turned off. Direct analysis of the presence of the Fe protein of nitrogenase in T. thiebautii by using a Western immunoblot procedure found a strong protein band present in samples collected after 0800 h through the late evening but little or no Fe protein in samples collected within the 2 to 4 h preceding dawn. We conclude that the diel cycle of NA in T. thiebautii results from de novo synthesis of nitrogenase each morning and from the inactivation and degradation of nitrogenase in the late afternoon and night.