Mannitol in six autotrophic stramenopiles and Micromonas

Mannitol plays a central role in brown algal physiology since it represents an important pathway used to store photoassimilate. Several specific enzymes are directly involved in the synthesis and recycling of mannitol, altogether forming the mannitol cycle. The recent analysis of algal genomes has allowed tracing back the origin of this cycle in brown seaweeds to a horizontal gene transfer from bacteria, and furthermore suggested a subsequent transfer to the green micro-alga Micromonas. Interestingly, genes of the mannitol cycle were not found in any of the currently sequenced diatoms, but were recently discovered in pelagophytes and dictyochophytes. In this study, we quantified the mannitol content in a number of ochrophytes (autotrophic stramenopiles) from different classes, as well as in Micromonas. Our results show that, in accordance with recent observations from EST libraries and genome analyses, this polyol is produced by most ochrophytes, as well as the green alga tested, although it was found at a wide range of concentrations. Thus, the mannitol cycle was probably acquired by a common ancestor of most ochrophytes, possibly after the separation from diatoms, and may play different physiological roles in different classes.Key words: algae, stramenopiles, mannitol cycle, primary metabolism, osmotic stress, evolutionBrown algae produce mannitol directly from the photoassimilate fructose-6-phosphate. Its metabolism occurs through the mannitol cycle, which involves four enzymatic reactions: (1) the reduction of fructose-6-phosphate (F6P) to mannitol-1-phosphate (M1P) via the activity of an M1P dehydrogenase (M1PDH); (2) the production of mannitol from M1P via an M1P phosphatase (M1Pase); (3) the oxidation of mannitol via the activity of a mannitol-2-dehydrogenase (M2DH) yielding fructose; and (4) the phosphorylation of fructose yielding F6P and involving a hexokinase (HK).^1,2 The first completed draft of a brown algal genome enabled the identification of candidate genes for each of these steps.³ As these genes were not found in the genomes of the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum, mannitol metabolism in stramenopiles was considered a trait typical for brown algae. The corresponding genes were thought to have been acquired horizontally from bacteria and subsequently transferred to some green algae.⁴ Recently, however, homologs of several genes of the cycle were also found in the genome of the pelagophyte Aureococcus anophagefferens⁵ and an EST library produced for the dictyochophyte Pseudochattonella farcimen (Dittami et al. personal communication). These observations prompted us to examine the presence of mannitol in a range of strains covering different classes of autotrophic stramenopiles (ochrophytes). In addition, because of the identification of genes encoding enzymes for the production of mannitol through the mannitol cycle in the green alga Micromonas, one strain of this genus was also included in our analysis.     

NADPH phagocyte oxidase knockout mice control Trypanosoma cruzi proliferation, but develop circulatory collapse and succumb to infection

^•NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91^{phox −/−} or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with ^•NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.     

高温油藏内源微生物及其提高采收率潜力研究

大港孔店油田油藏特征、流体和微生物性质分析结果表明, 属于高温生态环境, 地层水矿化度较低, 氮、磷浓度低, 而且缺乏电子受体, 主要的有机物来源是油气。油田采用经过除油处理的油藏产出水回注方式开发, 油层中存在的微生物类型主要是厌氧嗜热菌, 包括发酵菌(102个/mL~105个/mL), 产甲烷菌(103个/mL); 好氧菌主要存在于注水井周围。硫酸盐还原菌(SRB)还原速率0.002 mg S2-/(L·d) ~18.9 mg S2-/(L·d), 产甲烷菌产甲烷速率0.012 mgCH4/(L·d)~16.2 mgCH4/(L·d)。好氧菌能够氧化油形成生物质, 部分氧化产物为挥发性脂肪酸和表面活性剂。产甲烷菌在油氧化菌液体培养基中产生CH4, CO2为好氧微生物和厌氧微生物的共同代谢产物。这些产物具有提高原油流动性的作用。用示踪剂研究了注入水渗流方向。通过综合分析, 油藏微生物具有较大的潜力, 基于激活油层菌的提高采收率方法在该油田是可行的。     

Evolutionary trend in feeding habits of <Emphasis Type="Italic">Girella</Emphasis> (Perciformes: Girellidae)

Although some Girella species are herbivorous, having basically tricuspid teeth, some are omnivorous. To determine the evolutionary trends in feeding habits of Girella, the phylogenetic relationships of several species of Girella were estimated by partially sequencing the mitochondrially encoded NADH dehydrogenase subunit 2 gene, and the dentition and adductor mandibulae complex of each species were examined. The cladogram determined from the mitochondrial DNA analysis indicated that multiple tooth-rows containing incisor-like teeth existed in adults of the ancestral species of Girella, species with a single tooth-row of tricuspid teeth in the adult stage having diverged subsequently on several occasions. The tendinous connections between each section of the adductor mandibulae complex are believed to have been simple in the ancestral species, more complicated connections also having diverged later on several occasions. Multiple tooth-rows containing incisor-like teeth and the simple adductor mandibulae complex are deduced as adaptations to herbivory; on the other hand, a single tooth-row of tricuspid teeth and the complicated adductor mandibulae complex are deduced as adaptations to omnivory. Therefore, the ancestral species of Girella is suggested as having been adapted to herbivory, with species adapted to omnivory having diverged on several subsequent occasions.     

Does epigenetic polymorphism contribute to phenotypic variances in <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.?

Background  

There is a growing interest in Jatropha curcas L. (jatropha) as a biodiesel feedstock plant. Variations in its morphology and seed productivity have been well documented. However, there is the lack of systematic comparative evaluation of distinct collections under same climate and agronomic practices. With the several reports on low genetic diversity in jatropha collections, there is uncertainty on genetic contribution to jatropha morphology.     

RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome‐like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin‐1 interacted directly with the RuvbL1 barrel‐like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.     

Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi

The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha- mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.