Mammalian Mitochondrial DNA Replication Intermediates Are Essentially Duplex but Contain Extensive Tracts of RNA/DNA Hybrid

We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.     

The Recombinant Amyloid-β Peptide Aβ1-42 Aggregates Faster and Is More Neurotoxic than Synthetic Aβ1-42

Aggregation of the amyloid-β (Aβ) peptide is considered a central event in the pathogenesis of Alzheimer's disease (AD). In order to bypass methodological bias related to a variety of impurities commonly present in typical preparations of synthetic Aβ, we developed a simple, generally applicable method for recombinant production of human Aβ and Aβ variants in Escherichia coli that provides milligram quantities of Aβ in very high purity and yield. Amyloid fibril formation in vitro by human Aβ1-42, the key amyloidogenic Aβ species in AD, was completed threefold faster with recombinant Aβ1-42 compared to synthetic preparations. In addition, recombinant Aβ1-42 was significantly more toxic to cultured rat primary cortical neurons, and it was more toxic in vivo, as shown by strongly increased induction of abnormal phosphorylation of tau and tau aggregation into neurofibrillary tangles in brains of P301L tau transgenic mice. We conclude that even small amounts of impurities in synthetic Aβ—including a significant fraction of racemized peptides that cannot be avoided due to the technical limitations of peptide synthesis—prevent or slow Aβ incorporation into the regular quaternary structure of growing β-amyloid fibrils. The results validate the use of recombinant Aβ1-42 for both in vitro and in vivo studies addressing the mechanisms underlying Aβ aggregation and its related biological consequences for the pathophysiology, therapy, and prevention of AD.     

Combination of the Loss of cmnmU34 with the Lack of sU34 Modifications of tRNA, tRNA, and tRNA Altered Mitochondrial Biogenesis and Respiration

Yeast Saccharomyces cerevisiae MTO2, MTO1, and MSS1 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of carboxymethylaminomethyl (cmnm)⁵s²U₃₄ in mitochondrial tRNA^Lys, tRNA^Glu, and tRNA^Gln. In fact, Mto1p and Mss1p are involved in the biosynthesis of the cmnm⁵ group (cmnm⁵U₃₄), while Mto2p is responsible for the 2-thiouridylation (s²U₃₄) of these tRNAs. Previous studies showed that partial modifications at U₃₄ in mitochondrial tRNA enabled mto1, mto2, and mss1 strains to respire. In this report, we investigated the functional interaction between MTO2, MTO1, and MSS1 genes by using the mto2, mto1, and mss1 single, double, and triple mutants. Strikingly, the deletion of MTO2 was synthetically lethal with a mutation of MSS1 or deletion of MTO1 on medium containing glycerol but not on medium containing glucose. Interestingly, there were no detectable levels of nine tRNAs including tRNA^Lys, tRNA^Glu, and tRNA^Gln in mto2/mss1, mto2/mto1, and mto2/mto1/mss1 strains. Furthermore, mto2/mss1, mto2/mto1, and mto2/mto1/mss1 mutants exhibited extremely low levels of COX1 and CYTB mRNA and 15S and 21S rRNA as well as the complete loss of mitochondrial protein synthesis. The synthetic enhancement combinations likely resulted from the completely abolished modification at U₃₄ of tRNA^Lys, tRNA^Glu, and tRNA^Gln, caused by the combination of eliminating the 2-thiouridylation by the mto2 mutation with the absence of the cmnm⁵U₃₄ by the mto1 or mss1 mutation. The complete loss of modifications at U₃₄ of tRNAs altered mitochondrial RNA metabolisms, causing a degradation of mitochondrial tRNA, mRNA, and rRNAs. As a result, failures in mitochondrial RNA metabolisms were responsible for the complete loss of mitochondrial translation. Consequently, defects in mitochondrial protein synthesis caused the instability of their mitochondrial genomes, thus producing the respiratory-deficient phenotypes. Therefore, our findings demonstrated a critical role of modifications at U₃₄ of tRNA^Lys, tRNA^Glu, and tRNA^Gln in maintenance of mitochondrial genome, mitochondrial RNA stability, translation, and respiratory function.     

Adapter-Directed Display: A Modular Design for Shuttling Display on Phage Surfaces

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.     

Concomitant polymorphism: Crystallising dichloro-bis(2,4-lutidine)-zinc as both chiral and racemic phases

The crystallisation of dichloro-bis(2,4-lutidine)-zinc from various solvents (e.g. ethanol, THF and 2,4-lutidine) has been investigated and two phases were isolated. The structures of both phases were determined by single crystal X-ray diffraction and both types of crystals were found to be composed of conformationally chiral molecules. One phase (α-1) is racemic and crystallises in space group P2₁/c, while the other phase (β-1) crystallises in the enantiomorphous space group P4₁2₁2 with a low Flack parameter. In a few cases the chiral and racemic phases crystallised concomitantly; this phenomenon is rare and can be useful in the development of tools for the prediction of crystal structures.     

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl‐xL overexpression

Bcl‐x_L, a member of the Bcl‐2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl‐x_L overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐x_L expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) and MS analysis. The consequences of Bcl‐x_L overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐x_L overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif‐containing GTPase‐activating protein 1), cell proliferation (dihydrolipoamide‐S‐acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl‐x_L not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects. Biotechnol. Bioeng. 2010; 105: 358–367. © 2009 Wiley Periodicals, Inc.     

Synthesis and characterization of quaternized β-chitin

Water-soluble 2′-O-hydroxypropyltrimethylammoniumchitin chloride (2′-O-HTACCt) was prepared directly from β-chitin and 3-chloro-2-hydroxypropyltrimethylammonium chloride (CTA) in basic medium. The effect of alkali concentration, reaction temperature, reaction time, and dosage of CTA on yield and degree of substitution (DS) of 2′-O-HTACCt were studied. These quaternized chitin derivatives were characterized by FTIR and ¹H NMR spectroscopy, conductometric titration, and elemental analysis methods. Research results indicate that β-chitin can react directly with CTA to produce a water-soluble 2′-O-HTACCt derivative with a high DS. The optimal preparation conditions were determined to be 35-40 wt % (aq NaOH), 40 °C (reaction temperature), 6 h (reaction time), and 4 (molar ratio of CTA to β-chitin unit).     

Enhanced activity and stability of cellobiase (β-glucosidase: EC 3.2.1.21) produced in the presence of 2-deoxy-d-glucose from the fungus Termitomyces clypeatus

Generally less glycosylation or deglycosylation has a detrimental effect on enzyme activity and stability. Increased production and secretion of cellobiase was earlier obtained in the presence of the glycosylation inhibitor 2-deoxy-d-glucose in filamentous fungus Termitomyces clypeatus [Mukherjee, S.; Chowdhury, S.; Ghorai, S.; Pal, S.; Khowala, S. Biotechnol. Lett.2006, 28, 1773-1778]. In this study the enzyme was purified from the culture medium by ultrafiltration and gel-permeation, ion-exchange and high-performance liquid chromatography, and its catalytic activity was six times higher compared to the control enzyme. K_m and V_max of the purified enzyme were measured as 0.187 mM and 0.018 U mg⁻¹, respectively, using pNPG as the substrate. The enzyme had temperature and pH optima at 45 °C and pH 5.4, respectively, and retained full activity in a pH range of 5-8 and temperatures of 30-60 °C. Interestingly less glycosylated cellobiase was resistant towards proteolytic as well as endoglycosidase-H digestion and showed higher stability than native enzyme due to increased aggregation of the protein. The enzyme also showed higher specific activity in the presence of cellobiose and pNPG and less susceptibility towards salts and different chemical agents. The β-glucosidase can be considered as a potentially useful enzyme in various food-processing, pharmaceutical and fermentation industries.     

Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint

Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18→E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.     

Mcl-1 fragment induces apoptosis through direct interaction with Bax

Mcl-1 full-length (Mcl-1^1-350), a tightly regulated protein, plays an important role in protecting cells against apoptosis. Cleavage of Mcl-1 at Asp127 by caspase (Mcl-1^C1) contributes to the regulation of Mcl-1 expression, but its pro-apoptotic function remains controversial. Here, we reported that Mcl-1^128-350 expression induced caspase-dependent apoptosis. We demonstrated that Mcl-1^128-350 but not Mcl-1^1-350 interacts with Bax. This interaction required an intact BH3 Mcl-1^128-350 domain and leads to Bax activation and translocation to mitochondria. The silencing of Bax, but not of Bak, prevented Mcl-1^128-350 induced apoptosis. In conclusion, Mcl-1^128-350 exerts a pro-apoptotic function governed by its capacity to interact with Bax.

Structured summary

MINT-7306752: Mcl-1 (uniprotkb:Q07820) physically interacts (MI:0915) with BAK (uniprotkb:Q16611) by anti tag coimmunoprecipitation (MI:0007)MINT-7306728: Mcl-1 (uniprotkb:Q07820) physically interacts (MI:0914) with BAX (uniprotkb:Q07812) and BAK (uniprotkb:Q16611) by anti tag coimmunoprecipitation (MI:0007)MINT-7307171: F1 ATPase (uniprotkb:Q5TC12), Mcl-1 (uniprotkb:Q07820) and BAX (uniprotkb:Q07812) colocalize (MI:0403) by cosedimentation through density gradients (MI:0029)