Molecular mechanism of glucose-6-phosphate utilization in the dinoflagellate Karenia mikimotoi

Phosphorus (P) is an essential nutrient for marine phytoplankton as for other living organisms, and the preferred form, dissolved inorganic phosphate (DIP), is often quickly depleted in the sunlit layer of the ocean. Phytoplankton have developed mechanisms to utilize organic forms of P (DOP). Hydrolysis of DOP to release DIP by alkaline phosphatase is believed to be the most common mechanism of DOP utilization. Little effort has been made, however, to understand other potential molecular mechanisms of utilizing different types of DOP. This study investigated the bioavailability of glucose-6-phosphate (G6P) and its underlying molecular mechanism in the dinoflagellate Karenia mikimotoi. Suppression Subtraction Hybridization (SSH) was used to identify genes up- and down-regulated during G6P utilization compared to DIP condition. The results showed that G6P supported the growth and yield of K. mikimotoi as efficiently as DIP. Neither DIP release nor AP activity was detected in the cultures grown in G6P medium, however, suggesting direct uptake of G6P. SSH analysis and RT-qPCR results showed evidence of metabolic modifications, particularly that mitochondrial ATP synthase f1 gamma subunit and thioredoxin reductase were up-regulated while diphosphatase and pyrophosphatase were down-regulated in the G6P cultures. All the results indicate that K. mikimotoi has developed a mechanism other than alkaline phosphatase to utilize G6P.     

Species-dependent effects of seawater acidification on alkaline phosphatase activity in dinoflagellates

Increases of atmospheric CO₂ cause ocean acidification (OA) and global warming, the latter of which can stratify the water column and impede nutrient supply from deep water. Phosphorus (P) is an essential nutrient for phytoplankton to grow. While dissolved inorganic phosphorus (DIP) is the preferred form of P, phytoplankton have evolved alkaline phosphatase (AP) to utilize dissolved organic phosphorus (DOP) when DIP is deficient. Although the function of AP is known to require pH > 7, how OA affects AP activity and hence the capacity of phytoplankton to utilize DOP is poorly understood. Here, we examined the effects of pH conditions (5.5–11) on AP activity from six species of dinoflagellates, an important group of marine phytoplankton. We observed a general pattern that AP activity declined sharply at pH 5.5, peaked between pH 7 and 8, and dropped at pH > 8. However, our data revealed remarkable interspecific variations in optimal pH and niche breadth of pH. Among the species examined, Fugacium kawagutii and Prorocentrum cordatum had an optimal pH at 8, and Alexandrium pacificum, Amphidinium carterae, Effrenium voratum, and Karenia mikimotoi showed an optimal pH of 7. However, whereas A. pacificum and K. mikimotoi had the broadest pH niche for AP (7–10) and F. kawagutii the second (8–10), Am. carterae, E. voratum, and P. cordatum exhibited a narrow pH range. The response of Am. carterae AP to pH changes was verified using purified AP heterologously expressed in Escherichia coli. These results in concert suggest OA will likely differentially impact the capacity of different phytoplankton species to utilize DOP in the projected more acidified and nutrient-limited future ocean.     

Degrading seagrass (<Emphasis Type="Italic">Posidonia oceanica</Emphasis>) ecosystems: a source of dissolved matter in the Mediterranean

Diurnal variation of dissolved oxygen (DO), organic and inorganic carbon (DOC, DIC), nitrogen (DON, DIN), and phosphorus (DOP, DIP) flux across the sediment–water interface was assessed in fish farm impacted and pristine seagrass (Posidonia oceanica) meadows in the Aegean Sea (Greece). DIC consumption decreased by 52% and DO production decreased by 60% in the light, suggesting reduced photosynthetic performance of the plant community under the fish cages probably due to organic matter loading. In light there was 4 and 15 times higher release of dissolved inorganic and organic matter, respectively, compared to dark incubations under the cages, indicating that fish farming impact is more intense during daytime. DO was taken up, while DIC was released in the dark in both stations, representing a direct measure of mineralization. Dissolved inorganic matter flux (as the sum of DIN and DIP fluxes) was positively related to DIC flux, rendering mineralization as the main driver of nutrient flux under the cages. On average, the impacted meadow released DIN and DIP both in light and dark, while efflux of dissolved organic matter (as the sum of DOC, DON, and DOP fluxes) increased by 132% in the light and by 21% in the dark, implying that the degrading seagrass meadow is a source of dissolved matter to the surrounding water. Shoot density and leaf production were negatively correlated with both diel DIN and DIP fluxes, showing that meadow regression is accompanied by DIN and DIP release from the sediment. Hence, nutrient efflux can adequately illustrate meadow deterioration and, therefore, can be used as indicator of P. oceanica community health.     

Preferential utilization of inorganic polyphosphate over other bioavailable phosphorus sources by the model diatoms Thalassiosira spp.

Polyphosphates and phosphomonoesters are dominant components of marine dissolved organic phosphorus (DOP). Collectively, DOP represents an important nutritional phosphorus (P) source for phytoplankton growth in the ocean, but the contribution of specific DOP sources to microbial community P demand is not fully understood. In a prior study, it was reported that inorganic polyphosphate was not bioavailable to the model diatoms Thalassiosira weissflogii and Thalassiosira pseudonana. However, in this study, we show that the previous finding was a misinterpretation based on a technical artefact of media preparation and that inorganic polyphosphate is actually widely bioavailable to Thalassiosira spp. In fact, orthophosphate, inorganic tripolyphosphate (3polyP), adenosine triphosphate (ATP) and adenosine monophosphate supported equivalent growth rates and final growth yields within each of four strains of Thalassiosira spp. However, enzyme activity assays revealed in all cultures that cell-associated hydrolysis rates of 3polyP were typically more than ~10-fold higher than degradation of ATP and the model phosphomonoester compound 4-methylumbelliferyl phosphate. These results build on prior work, which showed the preferential utilization of polyphosphates in the cell-free exudates of Thalassiosira spp., and suggest that inorganic polyphosphates may be a key bioavailable source of P for marine phytoplankton.     

Dissolved organic phosphorus utilization by the marine bacterium Ruegeria pomeroyi DSS-3 reveals chain length-dependent polyphosphate degradation

Dissolved organic phosphorus (DOP) is a critical nutritional resource for marine microbial communities. However, the relative bioavailability of different types of DOP, such as phosphomonoesters (P-O-C) and phosphoanhydrides (P-O-P), is poorly understood. Here we assess the utilization of these P sources by a representative bacterial copiotroph, Ruegeria pomeroyi DSS-3. All DOP sources supported equivalent growth by R. pomeroyi, and all DOP hydrolysis rates were upregulated under phosphorus depletion (−P). A long-chain polyphosphate (45polyP) showed the lowest hydrolysis rate of all DOP substrates tested, including tripolyphosphate (3polyP). Yet the upregulation of 45polyP hydrolysis under −P was greater than any other substrate analyzed. Proteomics revealed three common P acquisition enzymes potentially involved in polyphosphate utilization, including two alkaline phosphatases, PhoD and PhoX, and one 5′-nucleotidase (5′-NT). Results from DOP substrate competition experiments show that these enzymes likely have broad substrate specificities, including chain length-dependent reactivity toward polyphosphate. These results confirm that DOP, including polyP, are bioavailable nutritional P sources for R. pomeroyi, and possibly other marine heterotrophic bacteria. Furthermore, the chain-length dependent mechanisms, rates and regulation of polyP hydrolysis suggest that these processes may influence the composition of DOP and the overall recycling of nutrients within marine dissolved organic matter.     

Lake‐type‐specific seasonal patterns of nutrient limitation in German lakes,with target nitrogen and phosphorus concentrations for good ecological status

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Effects of supplementation with free glutamine and the dipeptide alanyl‐glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise

In this study, we investigated the effect of the supplementation with the dipeptide L ‐alanyl‐L ‐glutamine (DIP) and a solution containing L ‐glutamine and L ‐alanine on plasma levels markers of muscle damage and levels of pro‐inflammatory cytokines and glutamine metabolism in rats submitted to prolonged exercise. Rats were submitted to sessions of swim training for 6 weeks. Twenty‐one days prior to euthanasia, the animals were supplemented with DIP (n = 8) (1.5 g.kg^?1), a solution of free L ‐glutamine (1 g.kg^?1) and free L ‐alanine (0.61 g.kg^?1) (G&A, n = 8) or water (control (CON), n = 8). Animals were killed at rest before (R), after prolonged exercise (PE—2 h of exercise). Plasma concentrations of glutamine, glutamate, tumour necrosis factor‐α (TNF‐α), prostaglandin E2 (PGE2) and activity of creatine kinase (CK), lactate dehydrogenase (LDH) and muscle concentrations of glutamine and glutamate were measured. The concentrations of plasma TNF‐α, PGE2 and the activity of CK were lower in the G&A‐R and DIP‐R groups, compared to the CON‐R. Glutamine in plasma (p < 0.04) and soleus muscle (p < 0.001) was higher in the DIP‐R and G&A‐R groups relative to the CON‐R group. G&A‐PE and DIP‐PE groups exhibited lower concentrations of plasma PGE2 (p < 0.05) and TNF‐α (p < 0.05), and higher concentrations of glutamine and glutamate in soleus (p < 0.001) and gastrocnemius muscles (p < 0.05) relative to the CON‐PE group. We concluded that supplementation with free L ‐glutamine and the dipeptide LL ‐alanyl‐LL ‐glutamine represents an effective source of glutamine, which may attenuate inflammation biomarkers after periods of training and plasma levels of CK and the inflammatory response induced by prolonged exercise. Copyright © 2009 John Wiley & Sons, Ltd.     

The caveolin‐3 P104L mutation in LGMD‐1C patients inhibits non‐insulin‐stimulated glucose metabolism and growth but promotes myocyte proliferation

The caveolin‐3 (CAV3) protein is known to be specifically expressed in various myocytes, and skeletal muscle consumes most of the blood glucose as an energy source to maintain normal cell metabolism and function. The P104L mutation in the coding sequence of the human CAV3 gene leads to autosomal dominant disease limb‐girdle muscular dystrophy type 1C (LGMD‐1C). We previously reported that C2C12 cells transiently transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis after insulin stimulation. The present study aimed to examine whether the P104L mutation affects C2C12 cell glucose metabolism, growth, and proliferation without insulin stimulation. C2C12 cells stably transfected with CAV3‐P104L were established, and biochemical assays, western blot analysis and confocal microscopy were used to observe glucose metabolism as well as cell growth and proliferation and to determine the effect of the P104L mutation on the PI3K/Akt signaling pathway. Without insulin stimulation, C2C12 cells stably transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis, decreased CAV3 expression and reduced localization of CAV3 and GLUT4 on the cell membrane. The P104L mutant significantly reduced the cell diameters, but accelerated cell proliferation. Akt phosphorylation was inhibited, and protein expression of GLUT4, p‐GSK3β, and p‐p70s6K, which are molecules downstream of Akt, was significantly decreased. The CAV3‐P104L mutation inhibits glycometabolism and cell growth but accelerates C2C12 cell proliferation by reducing CAV3 protein expression and cell membrane localization, which may contribute to the pathogenesis of LGMD‐1C.     

Fates of Evolutionarily Distinct,Plastid‐type Glyceraldehyde 3‐phosphate Dehydrogenase Genes in Kareniacean Dinoflagellates

The ancestral kareniacean dinoflagellate has undergone tertiary endosymbiosis, in which the original plastid is replaced by a haptophyte endosymbiont. During this plastid replacement, the endosymbiont genes were most likely flowed into the host dinoflagellate genome (endosymbiotic gene transfer or EGT). Such EGT may have generated the redundancy of functionally homologous genes in the host genome—one has resided in the host genome prior to the haptophyte endosymbiosis, while the other transferred from the endosymbiont genome. However, it remains to be well understood how evolutionarily distinct but functionally homologous genes were dealt in the dinoflagellate genomes bearing haptophyte‐derived plastids. To model the gene evolution after EGT in plastid replacement, we here compared the characteristics of the two evolutionally distinct genes encoding plastid‐type glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in Karenia brevis and K. mikimotoi bearing haptophyte‐derived tertiary plastids: “gapC1h” acquired from the haptophyte endosymbiont and “gapC1p” inherited from the ancestral dinoflagellate. Our experiments consistently and clearly demonstrated that, in the two species examined, the principal plastid‐type GAPDH is encoded by gapC1h rather than gapC1p. We here propose an evolutionary scheme resolving the EGT‐derived redundancy of genes involved in plastid function and maintenance in the nuclear genomes of dinoflagellates that have undergone plastid replacements. Although K. brevis and K. mikimotoi are closely related to each other, the statuses of the two evolutionarily distinct gapC1 genes in the two Karenia species correspond to different steps in the proposed scheme.     

Insulin‐Induced Ectodomain Shedding of Heparin‐Binding Epidermal Growth Factor‐Like Growth Factor in Adipocytes In Vitro

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is synthesized as a type I transmembrane protein, which is proteolytically cleaved to release a soluble form via members of the a disintegrin and metalloproteinase (ADAM) family of proteolytic enzymes. This study was designed to elucidate the molecular mechanism underlying insulin‐induced HB‐EGF shedding in adipocytes in vitro. The 3T3‐L1 adipocytes with stable expression of alkaline phosphatase (AP)‐tagged proHB‐EGF (3T3‐L1/HB‐EGF‐AP adipocytes) were developed and AP activities of conditioned media were determined. Using 3T3‐L1/HB‐EGF‐AP adipocytes, we demonstrated that insulin induces HB‐EGF shedding in differentiated 3T3‐L1 adipocytes in a dose‐ and time‐dependent manner. There is no significant increase in insulin‐induced HB‐EGF shedding in undifferentiated 3T3‐L1 preadipocytes. Studies with metalloprotease inhibitors suggested that insulin‐induced HB‐EGF shedding in adipocytes is mediated at least in part via ADAM17. Treatment with recombinant HB‐EGF results in a dose‐ and time‐dependent increase in HB‐EGF shedding in adipocytes, which is significantly suppressed by pharmacologic blockade of ADAM17 (P < 0.01). Moreover, insulin‐induced HB‐EGF shedding in adipocytes is significantly inhibited by AG1478, an EGF receptor antagonist (P < 0.01). This study provides in vitro evidence that insulin induces HB‐EGF shedding in 3T3‐L1 adipocytes. Our data also suggest the role of ADAM17 in insulin‐induced HB‐EGF shedding in adipocytes.     

Effects of nutrients on growth of the red-tide dinoflagellate <Emphasis Type="Italic">Gyrodinium instriatum</Emphasis> Freudenthal et Lee and a possible link to blooms of this species

We investigated the impact of different nitrogen (N) and phosphorus (P) compounds and concentrations on the growth of Gyrodinium instriatum Freudenthal et Lee in laboratory experiments, and possible links to blooms of this species at Hakozaki Fishing Port, Fukuoka, Japan. G. instriatum utilized only inorganic N compounds as N sources for growth. In contrast, G. instriatum utilized many inorganic and organic phosphorus compounds. We used the Monod equation to describe the growth rate of G. instriatum in N- or P-limited batch cultures as a function of ambient nutrient concentrations. Kinetic growth parameters for maximum specific growth rate (μ_max) and half-saturation nutrient concentration (K _S) were 0.57 divisions d⁻¹ and 14.2 μmol l⁻¹, respectively, under N-limitation and 0.65 divisions d⁻¹ and 1.75 μmol l⁻¹, respectively, under P-limitation. Compared with these K _S values, all in situ average dissolved inorganic nitrogen (DIN) concentrations in Hakozaki Fishing Port were higher than K _S for N, but all in situ average dissolved inorganic phosphorus (DIP) concentrations were lower than K _S for P, whether a red tide occurred or not bloom. Moreover, average DIP concentration in April (a month critical to red-tide genesis) of 2004 (a non-red-tide year) was less than half those in 2002 and 2003 (red-tide years). Thus, differences in DIP concentrations may be an important factor controlling blooms of G. instriatum in Hakozaki Fishing Port.