From Waste to Plastic: Synthesis of Poly(3-Hydroxypropionate) in Shimwellia blattae

In recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16 were cloned and expressed in the 1,3-propanediol producer Shimwellia blattae. In a two-step cultivation process, recombinant S. blattae cells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process.     

Production of 3-hydroxypropionate homopolymer and poly(3-hydroxypropionate-co-4-hydroxybutyrate) copolymer by recombinant Escherichia coli

Conversion of 3-hydroxypropionate (3HP) from 1,3-propanediol (PDO) was improved by expressing dehydratase gene (dhaT) and aldehyde dehydrogenase gene (aldD) of Pseudomonas putida KT2442 under the promoter of phaCAB operon from Ralstonia eutropha H16. Expression of these genes in Aeromonas hydrophila 4AK4 produced up to 21 g/L 3HP in a fermentation process. To synthesize homopolymer poly(3-hydroxypropionate) (P3HP), and copolymer poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P3HP4HB), dhaT and aldD were expressed in E. coli together with the phaC1 gene encoding polyhydroxyalkanoate (PHA) synthase gene of Ralstonia eutropha, and pcs' gene encoding the ACS domain of the tri-functional propionyl-CoA ligase (PCS) of Chloroflexus aurantiacus. Up to 92 wt% P3HP and 42 wt% P3HP4HB were produced by the recombinant Escherichia coli grown on PDO and a mixture of PDO+1,4-butanediol (BD), respectively.     

Biosynthesis and Biodegradation of 3-Hydroxypropionate- Containing Polyesters

3-Hydroxypropionate (3HP) is an important compound in the chemical industry, and the polymerized 3HP can be used as a bioplastic. In this review, we focus on polyesters consisting of 3HP monomers, including the homopolyester poly(3-hydroxypropionate) and copolyesters poly(3-hydroxybutyrate-co-3-hydroxypropionate), poly(3-hydroxypropionate-co-3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate), poly(4-hydroxybutyrate-co-3-hydroxypropionate-co-lactate), and poly(3-hydroxybutyrate-co-3-hydroxypropionate-co-4-hydroxybutyrate-co-lactate). Homopolyesters like poly(3-hydroxybutyrate) are often highly crystalline and brittle, which limits some of their applications. The incorporation of 3HP monomers reduces the glass transition temperature, the crystallinity, and also, at up to 60 to 70 mol% 3HP, the melting point of the copolymer. This review provides a survey of the synthesis and physical properties of different polyesters containing 3HP.Bacterial polyhydroxyalkanoates (PHAs) are natural biodegradable thermoplastics produced by various microorganisms as intracellular energy and carbon storage compounds. PHAs have attracted increased attention as possible alternatives to petroleum-based polymers. They are biodegradable, insoluble in water, nontoxic, biocompatible, piezoelectric, thermoplastic, and/or elastomeric. These features make PHAs suitable for several applications in the packaging industry, medicine, pharmacy, agriculture, and food industry, as raw materials for the production of enantiomerically pure chemicals, and for the production of paints (2, 44). The best characterized PHA is poly(3-hydroxybutyrate) [poly(3HB)], which is synthesized by many bacteria (28, 31, 38, 41). Unfortunately, poly(3HB) is a highly crystalline and brittle polymer with a low elongation-to-break factor, which has prevented its use in a wide range of applications. To obtain bacterial PHAs with improved physical and mechanical properties, previous studies have demonstrated the biosynthesis of copolyesters consisting of 3-hydroxybutyrate (3HB) and a second constituent. Pathways for the biosynthesis of such PHA copolyesters, like poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)], poly(3-hydroxybutyrate-co-4-hydroxyvalerate) [poly(3HB-co-4HV)], and poly(3-hydroxybutyrate-co-3-hydroxypropionate) [poly(3HB-co-3HP)], occur naturally in many bacteria or have been engineered (5, 40).Some of these polyesters exhibit material characteristics comparable to those of petrochemical-derived polymers. However, in contrast to petrochemical-based polymers, PHAs are completely biodegradable to CO₂ and water. Another advantage is that they can be produced from renewable resources. Unfortunately, PHA production by bacterial fermentation is costly and, due to inefficient utilization of the resources, not necessarily environmentally convenient in all cases (13).An example of a bacterium-synthesized copolymer which is competitive with polymers produced from petrochemicals in bulk is poly(3HB-co-3HV). Cupriavidus necator (32), formerly Ralstonia eutropha or Alcaligenes eutrophus, accumulates this copolyester when fed with glucose and propionic acid in a phosphate-depleted batch culture (30). The physical properties of poly(3HB-co-3HV) resemble those of polyethylene and polypropylene (18). Poly(3HB-co-3HV) has been commercially produced through fermentation using a glucose-utilizing mutant of C. necator that requires cofeeding of propionic acid for 3-hydroxyvalerate formation. This polymer was sold by ICI (in 1983) under the trade name Biopol.In general, the morphology and several physical properties of copolymers strongly depend on their comonomer composition and sequence structure (20). A comonomer lowering the melting temperature, crystallinity, and fragility is 3-hydroxypropionate (3HP). 3HP is an industrially relevant product. The putative applications of 3HP are enormous. Among the possible uses as a monomer for (co)polymerization, it could be used as a precursor for the synthesis of other commercially valuable chemicals, like 1,3-propanediol, acrylic acid, or acrylamide (6).3HP is only produced as a homopolymer from an unrelated carbon source by metabolic engineering in recombinant Escherichia coli (3); alternatively, it is chemically synthesized via ring-opening polymerization of β-propiolactone (4, 15, 49). In this paper, we review the synthesis and physical properties of 3HP-containing copolymers and the preparation of the 3HP units (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Artificial pathways for poly(HA-co-3HP) accumulation from different carbon sources. Acc_Cn, acetyl-CoA carboxylase (C. necator); Aco_Cn, acetyl-CoA synthase (C. necator); Acs_Ca, 3HP-CoA synthase domain of propionyl-CoA synthase (C. aurantiacus); DhaB_Cb, glycerol dehydratase (C. butyricum); Mcr_Ca, malonyl-CoA reductase (C. aurantiacus); OrfZ_Ck, acetyl-CoA:4-hydroxybutyrate-CoA transferase (C. kluyveri); Pct_Cp, propionyl-CoA transferase (C. propionicum); PduP_Se, propionaldehyde dehydrogenase (Salmonella enterica serovar Typhimurium LT2); PhaC_Cn, PHA synthase (C. necator); PrpE_Se, propionyl-CoA synthetase (S. enterica). The indices (n) at the hydroxyalkanoate moieties indicate the presence of lactate (n = 0), 3-hydroxyalkanoates (n = 1), and 4-hydroxybutyrate (n = 4).     

Microbial production of 4-hydroxybutyrate,poly-4-hydroxybutyrate,and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by recombinant microorganisms

4-Hydroxybutyrate (4HB) was produced by Aeromonas hydrophila 4AK4, Escherichia coli S17-1, or Pseudomonas putida KT2442 harboring 1,3-propanediol dehydrogenase gene dhaT and aldehyde dehydrogenase gene aldD from P. putida KT2442 which are capable of transforming 1,4-butanediol (1,4-BD) to 4HB. 4HB containing fermentation broth was used for production of homopolymer poly-4-hydroxybutyrate [P(4HB)] and copolymers poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)]. Recombinant A. hydrophila 4AK4 harboring plasmid pZL-dhaT-aldD containing dhaT and aldD was the most effective 4HB producer, achieving approximately 4 g/l 4HB from 10 g/l 1,4-BD after 48 h of incubation. The strain produced over 10 g/l 4HB from 20 g/l 1,4-BD after 52 h of cultivation in a 6-L fermenter. Recombinant E. coli S17-1 grown on 4HB containing fermentation broth was found to accumulate 83 wt.% of intracellular P(4HB) in shake flask study. Recombinant Ralstonia eutropha H16 grew to over 6 g/l cell dry weight containing 49 wt.% P(3HB-13%4HB) after 72 h.     

Augmentation of H2 photoproduction in Rhodopseudomonas palustris by N-heterocyclic aromatic compounds

Increases of 23- (5.6 mmol acetylene reduced mg dry wt^–1) and 16- (4 mmol acetylene reduced mg dry wt^–1) fold in nitrogenase activity and 12- (671 l H₂ mg dry wt^–1 h^–1) and 6- (349 l mg dry wt^–1 h^–1) fold in H₂ photoproduction in Rhodopseudomonas palustris JA1 over 24 h were achieved with pyrazine 2-carboxylate (3 mM) and 3-picoline (3 mM), respectively, and were higher than earlier reports of enhancement (1.5 to 5- fold) in biological H₂ production using various alternative methods.     

Synthesis of poly(hydroxyalkanoates) by Escherichia coli expressing mutated and chimeric PHA synthase genes

Pseudomonas resinovorans phaC1 _Pre and phaC2 _Pre genes coding for poly(hydroxyalkanoate) (PHA) synthases were cloned by PCR and expressed in E. coli LS1298 (fadB). Repeat-unit composition analysis showed that -hydroxydecanoate (67–75 mol%) and -hydroxyoctanoate (25–33 mol%) are the major monomers of the PHA produced in cells grown on decanoate. Sequence analysis showed that the gene products of phaC1 _Pre and phaC2 _Pre had 61% identical (75% positive) amino-acid sequence matches, and both sequences contained a conserved /-hydrolase fold in the carboxy-terminal portion of the proteins. Switching the /-hydrolase folds of phaC1 _Pre and phaC2 _Pre yielded chimeric pha7 and pha8 genes that afforded PHA synthesis in E. coli LS1298. The repeat-unit compositions of PHA in cells containing pha7 and pha8 were similar to those found in transformants containing the parental genes. Deletion mutants of phaC1 _Pre and phaC2 _Pre that resulted in potential translational fusions also supported PHA synthesis with similar repeat-unit compositions. Chimeric genes obtained from the switching of fragments containing the /-hydrolase folds of phaC1 _Pre and Ralstonia eutropha phbC did not direct the synthesis of PHA in transformed cells.     

Protein composition of nuclear 14 S ribonucleoprotein particles containing poly (A)

Nuclear 14 S RNP particles containing poly (A) from Ehrlich ascites carcinoma cells and rat liver were purified by re-sedimentation in sucrose gradients, by Cs₂SO₄ density gradient centrifugation and by affinity chromatography on a poly (dT)-Sepharose column. Proteins of these RNP particles were electrophoresed in urea and SDS-polyacrylamide gels. RNP particles of ascites carcinoma cells contain two main bands having molecular weights of 51 000 and 69 000 daltons, respectively, and two or three minor components.     

Electrical Impedance Myography to Detect the Effects of Electrical Muscle Stimulation in Wild Type and Mdx Mice

Objective

Tools to better evaluate the impact of therapy on nerve and muscle disease are needed. Electrical impedance myography (EIM) is sensitive to neuromuscular disease progression as well as to therapeutic interventions including myostatin inhibition and antisense oligonucleotide-based treatments. Whether the technique identifies the impact of electrical muscle stimulation (EMS) is unknown.

Methods

Ten wild-type (wt) C57B6 mice and 10 dystrophin-deficient (mdx) mice underwent 2 weeks of 20 min/day EMS on left gastrocnemius and sham stimulation on the right gastrocnemius. Multifrequency EIM data and limb girth were obtained before and at the conclusion of the protocol. Muscle weight, in situ force measurements, and muscle fiber histology were also assessed at the conclusion of the study.

Results

At the time of sacrifice, muscle weight was greater on the EMS-treated side than on the sham-stimulated side (p = 0.018 for wt and p = 0.007 for mdx). Similarly, in wt animals, EIM parameters changed significantly compared to baseline (resistance (p = 0.009), reactance (p = 0.0003) and phase (p = 0.002); these changes were due in part to reductions in the EIM values on the EMS-treated side and elevations on the sham-simulated side. Mdx animals showed analogous but non-significant changes (p = 0.083, p = 0.064, and p = 0.57 for resistance, reactance and phase, respectively). Maximal isometric force trended higher on the stimulated side in wt animals only (p = 0.06). Myofiber sizes in wt animals were also larger on the stimulated side than on the sham-stimulated side (p = 0.034); no significant difference was found in the mdx mice (p = 0.79).

Conclusion

EIM is sensitive to stimulation-induced muscle alterations in wt animals; similar trends are also present in mdx mice. The mechanisms by which these EIM changes develop, however, remains uncertain. Possible explanations include longer-term trophic effects and shorter-term osmotic effects.     

Submitochondrial localization and characteristics of thymidine kinase molecular forms in parental and kinase-deficient hela cells

Although HeLa (BU25) cells are deficient in cytosol dT kinase activity, they contain two mitochondrial dT kinases with disc PAGE mobilities (R _m) of 0.4 and 0.6 and isoelectric points (pI) of 8.4 and 5.6, respectively. Mitochondrial extracts of parental HeLa S3 contain the two HeLa (BU25) activities, but also a cytosol-like enzyme (0.25 R _m, pI 9.8). The 0.6-R _m (pI 5.6) mitochondrial activity utilizes ribonucleoside 5′-triphosphates other than ATP (dATP) as phosphate donors and is sensitive to dCTP inhibition. The predominant HeLa S3 cytosol (0.25 R _m) enzyme and the 0.4 R _m mitochondrial enzymeefficiently utilize only ATP as a phosphate donor and are relatively insensitive to dCTP inhibition. Submitochondrial fractionation studies have shown that (1) 74–98% of the mitochondrial dT kinase is located in the matrix plus inner membrane fractions; (2) the matrix fraction has the highest specific activity, contains all the 0.6-R _m activity, most of the HeLa S3 0.25-R _m activity, and some 0.4-R _m activity; (3) the inner membrane fraction is the major site of the 0.4-R _m activity but the outer membrane fraction also contains the 0.4 R _m activity; and (4) all HeLa S3 submitochondrial fractions contain the 0.25-R _m dT kinase activity.     

Expression of the NAD-dependent FDH1 β-subunit from Methylobacterium extorquens AM1 in Escherichia coli and its characterization

The efficient regeneration of nicotinamide cofactors is an important process for industrial applications because of their high cost and stoichiometric requirements. In this study, the FDH1 β-subunit of NAD-dependent formate dehydrogenase from Methylobacterium extorquens AM1 was heterologously expressed in Escherichia coli. It showed water-forming NADH oxidase (NOX-2) activity in the absence of its α-subunit. The β-subunit oxidized NADH and generated NAD⁺. The enzyme showed a low NADH oxidation activity (0.28 U/mg enzyme). To accelerate electron transfer from the enzyme to oxygen, four electron mediators were tested; flavin mononucleotide, flavin adenine dinucleotide, benzyl viologen (BV), and methyl viologen. All tested electron mediators increased enzyme activity; addition of 250 μM BV resulted in the largest increase in enzyme activity (9.98 U/mg enzyme; a 35.6-fold increase compared with that in the absence of an electron mediator). Without the aid of an electron mediator, the enzyme had a substrate-binding affinity for NADH (K _m) of 5.87 μM, a turnover rate (k _cat) of 0.24/sec, and a catalytic efficiency (k _cat/K _m) of 41.31/mM/sec. The addition of 50 μM BV resulted in a 22.75-fold higher turnover rate (k _cat, 5.46/sec) and a 2.64-fold higher catalytic efficiency (k _cat/K _m, 107.75/mM/sec).     

Differential regulation of the high‐affinity choline transporter by wild‐type and Swedish mutant amyloid precursor protein

The high‐affinity choline transporter (CHT) is responsible for choline uptake into cholinergic neurons, with this being the rate‐limiting step for acetylcholine production. Altering CHT protein disposition directly impacts choline uptake activity and cholinergic neurotransmission. Amyloid precursor protein (APP) interacts with CHT proteins and increases their endocytosis from the cell surface. The goal of this study was to examine regulation of CHT trafficking and activity by wild‐type APP (APP_wt) and determine if this differs with Swedish mutant APP (APP_Swe) in SH‐SY5Y human neuroblastoma cells. APP_Swe differs from APP_wt in its trafficking from the cell surface through endosomes. We report for the first time that CHT interacts significantly less with APP_Swe than with APP_wt. Surprisingly, however, CHT cell surface levels and choline uptake activity are decreased to the same extent and CHT co‐localization to early endosomes increased similarly in cells expressing either APP_wt or APP_Swe. A critical observation is that CHT co‐immunoprecipitates with βCTF from APP_Swe‐expressing cells. We propose that decreased CHT function is mediated differently by APP_wt and APP_Swe; APP_wt interaction with CHT facilitates its endocytosis from the cell surface, whereas the effect of APP_Swe on CHT is mediated indirectly potentially by binding to the βCTF fragment or by Aβ released from cells.

     

A Non-canonical DNA Structure Enables Homologous Recombination in Various Genetic Systems

Homologous recombination, which is critical to genetic diversity, depends on homologous pairing (HP). HP is the switch from parental to recombinant base pairs, which requires expansion of inter-base pair spaces. This expansion unavoidably causes untwisting of the parental double-stranded DNA. RecA/Rad51-catalyzed ATP-dependent HP is extensively stimulated in vitro by negative supercoils, which compensates for untwisting. However, in vivo, double-stranded DNA is relaxed by bound proteins and thus is an unfavorable substrate for RecA/Rad51. In contrast, Mhr1, an ATP-independent HP protein required for yeast mitochondrial homologous recombination, catalyzes HP without the net untwisting of double-stranded DNA. Therefore, we questioned whether Mhr1 uses a novel strategy to promote HP. Here, we found that, like RecA, Mhr1 induced the extension of bound single-stranded DNA. In addition, this structure was induced by all evolutionarily and structurally distinct HP proteins so far tested, including bacterial RecO, viral RecT, and human Rad51. Thus, HP includes the common non-canonical DNA structure and uses a common core mechanism, independent of the species of HP proteins. We discuss the significance of multiple types of HP proteins.Homologous recombination (HR)² is essential for gametogenesis during meiosis and plays an important role in the generation of genetic diversity, a process that is critical for natural selection. A general HR intermediate is the heteroduplex joint, which is formed between a single-stranded (ss) DNA tail derived from a double-stranded break and a homologous double-stranded (ds) DNA by homologous pairing (HP) (1, 2) and subsequent strand exchange (3, 4). HP is a switch from parental dsDNA base pairs to recombinant base pairs involving the ssDNA and the complementary strand of the dsDNA, which form the core of the recombination intermediate, and strand exchange is the unidirectional replacement of a dsDNA strand by the incoming ssDNA. The RecA/Rad51 family of proteins, which include bacterial RecA, archaeal RadA/Rad51, eukaryotic Rad51, and meiosis-specific Dmc1, are essential for HR in their respective organisms, and these proteins can promote ATP-dependent HP and ATP hydrolysis-dependent strand exchange in vitro (see Refs. 5–10, for reviews). In HP, ATP-bound RecA first binds to ssDNA, and this ssDNA·RecA complex then interacts with dsDNA without homologous recognition. Within the RecA·ssDNA·dsDNA complex, a homologous region is identified (11). The base pair switch in HP is formally carried out by base rotation or base flipping (rotation around the base-sugar bond), either of which requires the expansion of the spaces between neighboring bases or base pairs (see Refs. 7 and 12).Electron microscopic studies have shown that RecA/Rad51 proteins form a well conserved right-handed helical filament around ssDNA or dsDNA (13–15). In the absence of ATP, these proteins assemble as a shorter inactive filament (helical pitch, ≈65–85 Å) (16, 17). In the presence of ATP (or ATPγS, a non-hydrolyzable ATP analogue), the filament adopts an extended active conformation with a helical pitch of ≈95 Å, and the contour length of ssDNA and dsDNA within the active filament is elongated to the same extent (13–15, 17, 18). This equalized elongation has been inferred to widen the spacing between bases of ssDNA and dsDNA equally in the nucleoprotein filament to facilitate the homologous alignment of both DNA substrates to achieve base pair switching (13). Previously, we analyzed the three-dimensional structure of the RecA·ssDNA complex in the presence of ATPγS by NMR, which showed that the axial rise per ssDNA base was extended to nearly 5 Å (19), and that the interconversion of sugar puckers induced horizontal base rotation (20). On the basis of these results, and as there was no other structural information at that time, we proposed a base rotation mechanism to explain the base pair switch in HP by assuming that the ssDNA and dsDNA were extended equally and uniformly (20). Recently, the crystal structure of the RecA·ssDNA complex has revealed a non-uniformly extended structure for the ssDNA (21). The crystal structure contains “a three-nucleotide segment” (triplet) region and “a long untwisted inter-nucleotide” (inter-triplet) region (see Fig. 4). However, it remains unclear which structure contributes to HP and how it does so.Open in a separate windowFIGURE 4.Comparison of the solution and crystal structures of ssDNA bound to RecA. A, superimposition of the solution structure of RecA-d(TACG) (19) (in magenta) and four DNA residues in the RecA₅-(dT)₁₅ crystal structure (21) (in cyan). The crystal structure is similar to the solution structure. D1, D2, and D3 indicate distances between adjacent bases (see also 18, 20). Actually, the dsDNA was shown to be untwisted (unwound) within the homology-independent RecA-ssDNA-dsDNA intermediate of HP described above (22, 23). HP mediated by RecA or Rad51 was shown to be extensively stimulated by negative supercoiling of the dsDNA substrate in vitro (24, 25). This is probably because negative supercoils in the dsDNA substrate would compensate for the positive supercoils generated by the untwisting for HP. Closed circular dsDNA isolated from living cells, including DNA from bacteria, nuclei, or mitochondria of eukaryotic cells, is similarly supercoiled. However, in vivo, the supercoils of cellular dsDNA are relaxed by nucleosome assembly in eukaryotic nuclei and by the binding of HU (26) and/or other DNA-binding proteins in bacteria. Thus, dsDNA in vivo is an unfavorable substrate for HP mediated by RecA/Rad51 family proteins.In mitochondria, which do not have RecA/Rad51 family proteins, negative supercoils are relaxed by the binding of TFAM (in mammals) or Abf2 (in yeast) (27, 28). In this in vivo dsDNA state, Mhr1, an ATP-independent HP protein required for mitochondrial HR in the budding yeast Saccharomyces cerevisiae (29) catalyzes HP without the net untwisting of dsDNA, i.e. Mhr1 catalyzes HP with relaxed closed circular dsDNA with similar efficiency as with dsDNA lacking topological constraints (linear dsDNA and closed circular dsDNA in the presence of a topoisomerase) (30). Furthermore, in contrast to what is observed for RecA/Rad51, Mhr1-catalyzed HP is prevented by negative dsDNA supercoiling. The absence of net untwisting of dsDNA appears at first glance to mean HP without the extension of the parental dsDNA. However, HP requires the expansion of inter-base pair spaces for base pair switching as described above, and thus we proposed that right-handed wrapping of dsDNA around Mhr1 with an extended and untwisted configuration allows base rotation for HP (30). However, it remained to be experimentally determined whether Mhr1 and RecA/Rad51 share a common or different mechanism for HP.In addition to Mhr1, several proteins that promote HP in vitro in the absence of nucleotide cofactors have been identified. These include the human (hs) Xrcc3·Rad51c/Rad51L2 complex (human Rad51 paralogues; 31), hsRad52 (32), Escherichia coli (ec) phage λ β-protein (33), ecRecT (a homologue of λ β-protein 34), ecRecO (35), and Ustilago maydis Brh2 (36). Some of these proteins are termed recombination mediators, but we refer to them as ATP-independent HP proteins for the purpose of this study (supplemental Fig. S1). In contrast to RecA/Rad51 family members, ATP-independent HP proteins, except for those in the Xrcc3·Rad51C complex, do not exhibit any amino acid sequence homology with RecA/Rad51 proteins or other ATP-independent HP proteins. In addition, ATP-independent HP proteins exhibit significantly different quaternary structures (31, 32, 36–40). The N-terminal domain of hsRad52 forms an undecameric ring around which ssDNA and/or dsDNA wrap(s) (32, 41), and the interaction of closed circular dsDNA with hsRad52 generates negative supercoils (41), whereas binding to RecA generates positive supercoils in this substrate. On the other hand, like RecA, RecT was shown to untwist dsDNA during HP (42). The binding of dsDNA to Mhr1 causes neither untwisting nor twisting (30). Thus, the properties of HP proteins vary considerably except for their HP activities.In this study, we questioned whether the extended structure of ssDNA as seen in the RecA·ssDNA complex is conserved among HP proteins, or whether each HP protein uses a different principle to promote HP. If the extended structure is a common determinant of HP, the different HP proteins are likely to use a common mechanism to promote HP, but their variation may reflect requirements for optimizing HP in different cellular environments. Thus, we focused on the structure of the HP protein-bound ssDNA, an HP intermediate. We determined the three-dimensional structures of ssDNA bound to Mhr1 and of three other evolutionarily distinct HP proteins, ecRecT (ATP-independent, from the λ-like cryptic prophage Rac of E. coli, involved in plasmid HR (43)), Thermus thermophilus (tt) RecO (ATP-independent HP protein in bacteria), and hsRad51 (ATP-dependent, human nuclear homologue of RecA) and compared them with ssDNA bound to ecRecA (E. coli, the prototype of the RecA family; supplemental Fig. S1). This is the first demonstration that diverse HP proteins, both ATP-dependent (RecA/Rad51) and ATP-independent (Mhr1, RecO, RecT), use the non-canonical extended DNA structure as a common intermediate for HP, and this suggests that they use a common mechanism for HP.     

Combined Effects of Lead and Acid Rain on Photosynthesis in Soybean Seedlings

To explore how lead (Pb) and acid rain simultaneously affect plants, the combined effects of Pb and acid rain on the chlorophyll content, chlorophyll fluorescence reaction, Hill reaction rate, and Mg²⁺-ATPase activity in soybean seedlings were investigated. The results indicated that, when soybean seedlings were treated with Pb or acid rain alone, the chlorophyll content, Hill reaction rate, Mg²⁺-ATPase activity, and maximal photochemical efficiency (F _v/F _m) were decreased, while the initial fluorescence (F ₀) and maximum quantum yield (Y) were increased, compared with those of the control. The combined treatment with Pb and acid rain decreased the chlorophyll content, Hill reaction rate, Mg²⁺-ATPase activity, F _v/F _m, and Y and increased F ₀ in soybean seedlings. Under the combined treatment with Pb and acid rain, the two factors showed additive effects on the chlorophyll content in soybean seedlings and exhibited antagonistic effects on the Hill reaction rate. Under the combined treatment with high-concentration Pb and acid rain, the two factors exhibited synergistic effects on the Mg²⁺-ATPase activity, F ₀, F _v/F _m, as well as Y. In summary, the inhibition of the photosynthetic process is an important physiological basis for the simultaneous actions of Pb and acid rain in soybean seedlings.     

Biosynthesis of cat hemoglobins: Translation of poly(A)-RNA from animals of various HbA/HbB phenotypes

The molecular basis for the genetic control of variable proportions of the two hemoglobins in domestic cat blood was investigated. Both major hemoglobins of cat blood, HbA (α₂β ₂ ^A ) and HbB (α₂β ₂ ^B ), were synthesized in an mRNA-dependent rabbit reticulocyte system using poly(A)-RNA from cat reticulocyte polysomes as the source of the message. The relative amounts of HbA and HbB synthesized in the system were a function of the HbA/HbB phenotype of the cat from which the reticulocytes and poly(A)-RNA were obtained. Higher ratios of HbA/HbB synthesis were found when the source of poly(A)-RNA was the polysomes from a 90/10 (HbA/HbB) phenotype than when it was from a 50/50 (HbA/HbB) phenotype. These results indicate that the variable proportions of HbA and HbB found in the blood of different members of the cat population result from the genetic control of the relative amounts of functional β^A and β^B mRNA.     

Isolation and identification of an endophytic fungus Pezicula sp. in Forsythia viridissima and its secondary metabolites

In a survey of endophytic fungal biodiversity, an antimicrobial endophytic isolate zjwcf069 was obtained from twigs of Forsythia viridissima, Zhejiang Province, Southeast China. Zjwcf069 was then identified as Pezicula sp. through combination of morphological and phylogenetic analysis based on ITS-rDNA. Zjwcf069 here represented the first endophytic fungus in Pezicula isolated from host F. viridissima. From the fermentation broth, four compounds were obtained through silica gel column chromatography and Sephadex LH-20 under the guide of bioassay. Their structures were elucidated by spectroscopic analysis as mellein (1), ramulosin (2), butanedioic acid (3), and 4-methoxy-1(3H)-isobenzofuranone (4). Compound 4 here stood for the very first time as natural product from microbes. In vitro antifungal assay showed that compound 1 displayed growth inhibition against 9 plant pathogenic fungi, especially Botrytis cinerea and Fulvia fulva with EC₅₀ values below 50 μg/mL. Endophytic fungi in medicinal plants were good resources for bioactive secondary metabolites.     

The effect of Sa and MiniSa plasmids on the induction of plant crown galls and hairy roots byAgrobacterium tumefaciens andAgrobacterium rhizogenes

The Inc-W group plasmid Sa or its derivative MiniSa were introduced into two strains ofAgrobacterium tumefaciens with Ti plasmids, one strain ofA. tumefaciens with the Ri plasmid and oneA. rhizogenes strain with the Ri plasmid. The effect was similar in allAgrobacterium strains. The pSa suppressed fully the virulence ofAgrobacterium strains (i.e. their ability to induce tumor growths - crown galls or hairj⁷ roots) inKalanchoe plants and carrot root slices. The MiniSa plasmid caused only a slight decrease of the frequency and size of tumor growths induced. The mechanism of suppression of virulence by the Sa plasmid inAgrobacterium tumefaciens andAgrobacterium rhizogenes seems to be similar.     

Expression of Transthyretin during bovine myogenic satellite cell differentiation

Adult myogenesis responsible for the maintenance and repair of muscle tissue is mainly under the control of myogenic regulatory factors (MRFs) and a few other genes. Transthyretin gene (TTR), codes for a carrier protein for thyroxin (T₄) and retinol binding protein bound with retinol in blood plasma, plays a critical role during the early stages of myogenesis. Herein, we investigated the relationship of TTR with other muscle-specific genes and report their expression in muscle satellite cells (MSCs), and increased messenger RNA (mRNA) and protein expression of TTR during MSCs differentiation. Silencing of TTR resulted in decreased myotube formation and decreased expression of myosin light chain (MYL2), myosin heavy chain 3 (MYH3), matrix gla protein (MGP), and voltage-dependent L type calcium channel (Cav1.1) genes. Increased mRNA expression observed in TTR and other myogenic genes with the addition of T₄ decreased significantly following TTR knockdown, indicating the critical role of TTR in T₄ transportation. Similarly, decreased expression of MGP and Cav1.1 following TTR knockdown signifies the dual role of TTR in controlling muscle myogenesis via regulation of T₄ and calcium channel. Our computational and experimental evidences indicate that TTR has a relationship with MRFs and may act on calcium channel and related genes.