Use of low nutrient enrichments to access novel amylase genes in silent diversity of thermophiles

By combining low nutrient enrichments and molecular methods, a high diversity of new amylase genes was detected in a neutral sulphide-rich hot spring in Iceland. Enrichments based on hot spring water and low concentrations of starch were used to select slow-growing, starch-degrading microorganisms. Six enrichments had in total 17 bacterial types detected by 16S rRNA analysis, mostly related to the Thermus-Deinococcus group, green non-sulphur bacteria, gram positives, and uncultivated new candidate divisions. No Archaea were found. The apparent 16S rRNA species composition of the enrichments was very different from that of the microbial mat in the same hot spring. DNA samples obtained from 4 enrichments and from hot spring biomass were screened by PCR for amylase genes in glycoside-hydrolase family 13. Degenerate primers, based on conserved amino acid sequences from multiplealignments of family 13, enabled the detection of 18 amylase sequence types in the enrichments, including -amylases, -glucosidases, 1,4--glucan branching enzymes, cyclomaltodextrin hydrolases, maltogenic amylases and neopullulanases, and unspecified family 13 glycoside-hydrolases. Only one unique neopullulanase sequence, also found in most of the enrichments, was detected in the hot spring biomass DNA. The results suggest that the enrichment method combined with sequence-based screening is an efficient way to access the silent, i.e. not detectable, gene diversity in natural environments.     

蛋白质的选择性降解机制——2004年诺贝尔化学奖部分工作介绍

如何识别和选择性降解蛋白质是细胞生命过程中的重要环节.泛素-蛋白酶体需能降解途径的发现,揭示了蛋白质在细胞内选择性降解的普遍方式.对于需要清除的蛋白质,通过其赖氨酸残基侧链ε-氨基连接多聚泛素链（降解标签）,继而在蛋白酶体中被降解.这种选择性降解机制对于维持蛋白质在细胞内含量的动态平衡起到了关键性作用.     

A novel thermotolerant methylotrophicBacillus sp. and its potential for use in single-cell protein production

A thermotolerant methylotrophicBacillus sp. (KISRI TM1A, NCIMB 40040), isolated from the Kuwaiti environment and belonging to the group II spore-forming, bacilli, could not be correlated with any knownBacillus sp. It may, therefore, be a new species. It grew at temperatures from 37° to 58°C from pH 6.5 to 9.0 and on methanol up to 40 g l^–1. It grew well in a chemostat. Its biomass yield coefficient was improved by about 30% by optimization of medium and growth conditions, reaching a maximum of 0.44g g^–1 at 45°C pH 6.8 to 7.0, dilution rate 0.25 h^–1 with methanol at 10 g l^–1. Average crude protein and amino acid content were 84% and 60%, respectively, and maximum productivity attained under laboratory conditions was 5.06 g l^–1h^–1. It was concluded that this strain has good potential for use in single-cell protein production.     

Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.     

Biochemical aspects of the developing and ripening banana

The peel and pulp of the banana fruit and the pseudostem were examined for glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT) and aldolase activities and protein, phenolics, chlorophyll and starch. The peel-pulp ratio at various stages of fruit development on the plant and in detached fruits showing incipient ripening were used as an index of the physiological age of the fruit. The enzymes exhibited maximum activity at a stage corresponding to the initiation of the climacteric. GPT level at this stage was higher than that of GOT. An initial increase in the protein content was followed by a decline in both peel and pulp, the level reaching a minimum in climacteric fruits. Astringency, measured in terms of total phenolics, decreased with development; in mature fruits, peel contained 4–5 × as much phenolics as pulp. Chlorophyll in mature fruits was 10 × higher than in young fruits and decreased in ripe fruits. The onset of ripening was attended with a pronounced decrease in the starch. The various analyses were carried out also on the pseudostem removed from the plant soon after flower formation.     

Perturbation of the lipid phase of a membrane is not involved in the modulation of MRP1 transport activity by flavonoids

The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.     

Peroxisomal Targeting of PTS2 Pre-Import Complexes in the Yeast Saccharomyces cerevisiae

Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.     

一株降解纤维素放线菌的分类研究

从高温稻草堆肥中分离到1株能降解纤维素的放线菌CN9,分别对其表型特征、化学特征和16SrRNA基因序列进行了分析,根据实验结果,CN9属于高温链霉菌。     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.