Transmission of mitochondrial DNA in pigs and progeny derived from nuclear transfer of Meishan pig fibroblast cells

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Here, we examined the transmission of mtDNA from NT pigs to their progeny. NT pigs were created by microinjection of Meishan pig fetal fibroblast nuclei into enucleated oocytes (maternal Landrace background). Transmission of donor cell (Meishan) mtDNA was analyzed using 4 NT pigs and 25 of their progeny by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, PCR-RFLP, and a specific PCR to detect Meishan mtDNA single nucleotide polymorphisms (SNP-PCR). In the blood and hair root of NT pigs, donor mtDNAs were not detected by PCR-SSCP and PCR-RFLP, but detected by SNP-PCR. These results indicated that donor mtDNAs comprised between 0.1% and 1% of total mtDNA. Only one of the progeny exhibited heteroplasmy with donor cell mtDNA populations, ranging from 0% to 44% in selected tissues. Additionally, other progeny of the same heteroplasmic founder pig were analyzed, and 89% (16/18) harbored donor cell mtDNA populations. The proportion of donor mtDNA was significantly higher in liver (12.9 +/- 8.3%) than in spleen (5.0 +/- 3.9%), ear (6.7 +/- 5.3%), and blood (5.8 +/- 3.7%) (P < 0.01). These results demonstrated that donor mtDNAs in NT pigs could be transmitted to progeny. Moreover, once heteroplasmy was transmitted to progeny of NT-derived pigs, it appears that the introduced mitochondrial populations become fixed and maternally-derived heteroplasmy was more readily maintained in subsequent generations.     

Protein tagging at rare codons is caused by tmRNA action at the 3' end of nonstop mRNA generated in response to ribosome stalling

It has been believed that protein tagging caused by consecutive rare codons involves tmRNA action at the internal mRNA site. We demonstrated previously that ribosome stalling either at sense or stop codons caused by certain arrest sequences could induce mRNA cleavage near the arrest site, resulting in nonstop mRNAs that are recognized by tmRNA. These findings prompted us to re-examine the mechanism of tmRNA tagging at a run of rare codons. We report here that either AGG or CGA but not AGA arginine rare-codon clusters inserted into a model crp mRNA encoding cAMP receptor protein (CRP) could cause an efficient protein tagging. We demonstrate that more than three consecutive AGG codons are needed to induce an efficient ribosome stalling therefore tmRNA tagging in our system. The tmRNA tagging was eliminated by overproduction of tRNAs corresponding to rare codons, indicating that a scarcity of the corresponding tRNA caused by the rare-codon cluster is an important factor for tmRNA tagging. Mass spectrometry analyses of proteins generated in cells lacking or possessing tmRNA encoding a protease-resistant tag sequence indicated that the truncation and tmRNA tagging occur within the cluster of rare codons. Northern and S1 analyses demonstrated that nonstop mRNAs truncated within the rare-codon clusters are detected in cells lacking tmRNA but not in cells expressing tmRNA. We conclude that a ribosome stalled by the rare codon induces mRNA cleavage, resulting in nonstop mRNAs that are recognized by tmRNA.     

Effects of the turn-back primer on intermediate product generation in isothermal DNA amplification

In DNA amplification, the initial step of copying a target sequence from the template DNA--the so-called intermediate product generation step--is very important. In examining the turn-back primer (TP)-dependent isothermal DNA amplification (TIA) method, we determined the actual time point of intermediate product generation by extrapolating dsDNA amplification curves. Our results indicate that intermediate product creation is the rate-limiting step in TIA, and good TP design is advantageous for improving the intermediate production process.     

Key aromatic residues at subsites + 2 and + 3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates

Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest k_cat/K_m for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k_cat/K_m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k_cat/K_m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k_cat/K_m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at + 2 and + 3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs.     

Molecular Basis for the Recognition of Long-chain Substrates by Plant α-Glucosidases

Sugar beet α-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher k_cat/K_m values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific α-glucosidases. To gain structural insights into the long-chain substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide acarbose was determined at 1.7 Å resolution. The active site pocket of SBG is formed by a (β/α)₈ barrel domain and a long loop (N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues (Phe-236 and Asn-237) in the N-loop are important for the long-chain specificity. Kinetic analysis of an Asn-237 mutant enzyme and a previous study of a Phe-236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicates that Phe-236 and Asn-237 guide the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (β/α)₈ barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis.     

Differential expression of co-signal molecules and migratory properties in four distinct subsets of migratory dendritic cells from the oral mucosa

Variations in co-signal ligand expression and cytokine production greatly influence the antigen-presenting properties of migrating DCs in regional lymph nodes (RLNs). Here we investigated DCs migrating from the oral mucosa using CD326 and CD103 antigens for discriminate CD207⁺ Langerhans cells (LCs) from CD207⁺ submucosal DCs (SMDCs). Similar to DCs migrating from the skin, we identified four distinct oral mucosal DC (OMDC) subsets, CD11c^hiCD207⁻CD103⁻CD326^intCD11b^hi (F1; resident CD11b^hi SMDCs), CD11c^int/loCD207^-CD103^-CD326^loCD11b^int/hi (F2; newly recruited blood-derived SMDCs), CD11c^int/loCD207⁺CD103⁺CD326^int/hiCD11b^lo (CD103⁺ F3; resident CD207⁺ SMDCs), and CD11c^int/loCD207⁺CD103^-CD326^int/hiCD11b^lo (CD103^- F3; resident LCs). F1 DCs migrated rapidly after fluorescein isothiocyanate (FITC) painting and expressed notably high levels of CD86, CD273, and CD274 at an earlier time point. In contrast, CD103⁻ LCs expressing the highest levels of the epithelial cell adhesion molecule CD326 accounted for a minor subset at the earlier time point, but increased slowly with CD103⁺CD207⁺ SMDCs. However, their expression of CD86, CD273, and CD274 was very limited. The delayed migration and limited induction of co-signal ligands suggest that roles of OMLCs are distinct from those of the other three DC subsets. The identification of distinct subsets of OMDCs in RLNs may benefit efforts to determine the functional specialization of each subset in T cell responses against orally administrated antigens.     

Egg-collection equipment for Liriomyza leafminers (Diptera: Agromyzidae)

Egg-collection equipment for Liriomyza leafminers was designed using a small glass tube with a glucose solution supported between two thin films. Adult female Liriomyza trifolii (Burgess), which were previously reared on kidney bean for 2–4 days, laid abundant eggs in 5 or 10% glucose solutions. Hatchability of the eggs in the solutions was greater than 80%. The equipment was used successfully to collect eggs from another leafminer species, Liriomyza sativae Blanchard.     

Regio- and stereoselective oxidation of propranolol enantiomers by human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19

Toxic and pharmacokinetic profiles of drug candidates are evaluated in vivo often using monkeys as experimental animals, and the data obtained are extrapolated to humans. Well understanding physiological properties, including drug-metabolizing enzymes, of monkeys should increase the accuracy of the extrapolation. The present study was performed to compare regio- and stereoselectivity in the oxidation of propranolol (PL), a chiral substrate, by cytochrome P450 2D (CYP2D) enzymes among humans, cynomolgus monkeys and marmosets. Complimentary DNAs encoding human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19 were cloned, and their proteins expressed in a yeast cell expression system. The regio- and stereoselective oxidation of PL enantiomers by yeast cell microsomal fractions were compared. In terms of efficiency of expression in the system, the holo-proteins ranked CYP2D6 ≒ CYP2D17 ? CYP2D19. This may be caused by the bulky side chain of the amino acid residue at position 119 (leucine for CYP2D19 vs. valine for CYP2D6 and CYP2D17), which can disturb the incorporation of the heme moiety into the active-site cavity. PL enantiomers were oxidized by all of the enzymes mainly into 4-hydroxyproranolol (4-OH-PL), followed by 5-OH-PL and N-desisopropylpropranolol (NDP). In the kinetic analysis, apparent K_m values were commonly in the μM range and substrate enantioselectivity of R-PL < S-PL was observed in both K_m and V_max values for the formation of the three metabolites from PL enantiomers. The activity to produce NDP tended to be higher for the monkey enzymes, particularly CYP2D17, than for the human enzyme. These results indicate that in the oxidation of PL enantiomers by CYP2D enzymes, stereoselectivity is similar but regioselectivity is different between humans and monkeys.