Site-specific genome integration in alphaproteobacteria mediated by TG1 integrase

The serine-type phage integrase is an enzyme that catalyzes site-specific recombination between two attachment sites of phage and host bacterial genomes (attP and attB, respectively) having relatively short but distinct sequences without host auxiliary factor(s). Previously, we have established in vivo and in vitro site-specific recombination systems based on the serine-type integrase produced by actinophage TG1 and determined the minimal sizes of attP _TG1 and attB _TG1 sites required for the in vitro TG1 integrase reaction as 43- and 39-bp, respectively. Here, DNA databases were surveyed by FASTA program with the authentic attB _TG1 sequence of Streptomyces avermitilis as a query. As a result, possible attB _TG1 sequences were extracted from genomes of bacterial strains belonging to Class Alphaproteobacteria in addition to those of Class Actinobacteria. Those sequences extracted with a high similarity score and high sequence identity (we took arbitrarily more than 80% identity) turned out to be located within a conserved region of dapC or related genes encoding aminotransferases and proved to be actually recognized as the cognate substrate of attP _TG1 site by the in vitro TG1 integrase assay. Furthermore, the possible attB _TG1 site of Rhodospirillum rubrum revealed to be used actually as a native (endogenous) attachment site for the in vivo TG1-based integration system. These features are distinct from other serine-type phage integrases and advantageous for a tool of genome technology in varied industrially important bacteria belonging to Class Alphaproteobacteria.     

Paternal uniparental disomy 14 and related disorders: Placental gene expression analyses and histological examinations

Although recent studies in patients with paternal uniparental disomy 14 [upd(14)pat] and other conditions affecting the chromosome 14q32.2 imprinted region have successfully identified underlying epigenetic factors involved in the development of upd(14)pat phenotype, several matters, including regulatory mechanism(s) for RTL1 expression, imprinting status of DIO3 and placental histological characteristics, remain to be elucidated. We therefore performed molecular studies using fresh placental samples from two patients with upd(14)pat. We observed that RTL1 expression level was about five times higher in the placental samples of the two patients than in control placental samples, whereas DIO3 expression level was similar between the placental samples of the two patients and the control placental samples. We next performed histological studies using the above fresh placental samples and formalin-fixed and paraffin-embedded placental samples obtained from a patient with a maternally derived microdeletion involving DLK1, the-IG-DMR, the MEG3-DMR and MEG3. Terminal villi were associated with swollen vascular endothelial cells and hypertrophic pericytes, together with narrowed capillary lumens. DLK1, RTL1 and DIO3 proteins were specifically identified in vascular endothelial cells and pericytes, and the degree of protein staining was well correlated with the expression dosage of corresponding genes. These results suggest that RTL1as-encoded microRNA functions as a repressor of RTL1 expression, and argue against DIO3 being a paternally expressed gene. Furthermore, it is inferred that DLK1, DIO3 and, specially, RTL1 proteins, play a pivotal role in the development of vascular endothelial cells and pericytes.     

Mechanism for folate-independent aldolase reaction catalyzed by serine hydroxymethyltransferase

Serine hydroxymethyltransferase catalyzes the cleavage of β-hydroxyamino acids into glycine and aldehydes in the absence of tetrahydrofolate. The enzyme accepts various β-hydroxyamino acids as the substrate of this reaction. The reaction rate varies depending on the substituent and stereochemistry at the Cβ atom: the erythro forms and the β-phenyl substituent are preferred over the threo forms and the β-methyl substituent, respectively. Although several mechanisms have been proposed, what determines the substrate preference remains unclear. We first performed quantum mechanical calculations to assess the validity of the reaction mechanisms. The results indicate that the retro-aldol mechanism starting with abstraction of the proton from the β-hydroxyl group is plausible. This also suggests that Cα-Cβ bond cleavage is the rate-limiting step. We next measured the dependence of the rate constants on temperature with four representative substrates and calculated the activation energies and pre-exponential factors from the Arrhenius plots. The activation energies of the erythro forms were lower than those of the threo forms. The β-phenyl substituent lowered the activation energy in the threo form, whereas it did not alter the activation energy but increased the pre-exponential factor in the erythro form. We present a unified model to explain the origin of the substituent and stereochemical preferences by combining the theoretical and experimental results. A possible biological role of the tetrahydrofolate-independent activity in thermophiles is also discussed.     

Genetic Diversity and Transmission Characteristics of Beijing Family Strains of Mycobacterium tuberculosis in Peru

Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs). They were also subjected to variable number tandem-repeat (VNTR) typing using an international standard set of 15 loci (15-MIRU-VNTR) plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10), which belongs to a modern subfamily of Beijing strains (178/198, 89.9%). Twelve strains belonged to the ancient subfamily (ST3 [n = 3], ST25 [n = 1], ST19 [n = 8]). Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3%) and a high recent transmission index (RTI_n−1 = 0.707). These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999.     

Comparative analysis of Japanese and foreign L-type BSE prions

L-type bovine spongiform encephalopathy (BSE) is an atypical form of BSE. To characterize the Japanese L-type BSE prion, we conducted a comparative study of the Japanese and foreign L-type BSE isolates. The L-type BSE isolates of Japan, Germany, France and Canada were intracerebrally inoculated into bovinized prion protein-overexpressing transgenic mice (TgBoPrP). All the examined L-type BSE isolates were transmitted to TgBoPrP mice, and no clear differences were observed in their biological and biochemical properties. Here, we present evidence that the Japanese and Canadian L-type BSE prions are identical to those from the European cases.Key words: prion, atypical BSE, L-type BSEBovine spongiform encephalopathy (BSE) is one of the transmissible spongiform encephalopathies (TSEs), or prion diseases, in cattle. TSE is characterized by spongiform changes in the central nervous system (CNS) and the accumulation of an abnormal prion protein (PrP^Sc) in the CNS.¹ PrP^Sc has been regarded as the major component of TSE pathogens.²BSE was detected in the UK in 1986,³ and subsequently spread to the other European countries, Japan and North America.^4–6 BSE is thought to be caused by a single prion strain, based on the analyses of its biological and biochemical characteristics.⁷ From 2003, however, several atypical neuropathological and molecular phenotypes of BSE (atypical BSE) have been detected in Japan, several European countries and North America.^6,8–17 Currently, based on the molecular size of the proteinase-digested non-glycosylated form of PrP^Sc, atypical BSE is classified into two groups (L-type and H-type).¹⁴L-type BSE cases have been identified in the European countries, including Italy, France, Germany, Netherland, Poland and in Canada and Japan.^8–15 Two L-type BSE cases have been identified in Japan. One case was detected in a healthy 23-mo-old Holstein steer (BSE/JP8),⁸ and the other was detected in a 14-y-old black Japanese beef cattle (BSE/JP24).⁹ The latter case was successfully transmitted to bovinized transgenic mice and cattle, and the biological and biochemical properties differed from that of classical BSE (C-BSE).^18,19 However, it is unclear whether Japanese L-type BSE prion is identical to that of L-type BSE isolates from other countries. To characterize the Japanese L-type BSE isolate, we performed a comparative study of the Japanese and foreign L-type BSE isolates.A transmission study using experimental animals is a useful approach for prion characterization. Therefore, we performed a transmission study of the L-type BSE isolates in bovinized prion protein (PrP)-overexpressing transgenic mice (TgBoPrP).²⁰ Brain samples of L-type BSE-affected cattle from Japan (BSE/JP24),⁹ France,¹⁰ Germany¹¹ and Canada¹² were used in this study. The brain homogenates were intracerebrally inoculated into TgBoPrP using previously described methods in reference ¹⁸. All animal experiments were reviewed by the Committee of the Ethics on Animal Experiment of the National Institute of Animal Health.All the examined L-type BSE isolates were transmitted to TgBoPrP, and the affected mice developed progressive neurological diseases. Japanese L-type BSE isolate-affected TgBoPrP exhibited a unique clinical sign, the circling behavior. The same phenotype was observed when TgBoPrP were inoculated with German, French and Canadian L-type BSE isolates. On the other hand, in the first passage the incubation period for the Japanese L-type BSE isolate was significantly different from that of the other L-type BSE isolates (

Kinetochore stretching inactivates the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) monitors the attachment of microtubules to the kinetochore and inhibits anaphase when microtubule binding is incomplete. The SAC might also respond to tension; however, how cells can sense tension and whether its detection is important to satisfy the SAC remain controversial. We generated a HeLa cell line in which two components of the kinetochore, centromere protein A and Mis12, are labeled with green and red fluorophores, respectively. Live cell imaging of these cells reveals repetitive cycles of kinetochore extension and recoiling after biorientation. Under conditions in which kinetochore stretching is suppressed, cells fail to silence the SAC and enter anaphase after a delay, regardless of centromere stretching. Monitoring cyclin B levels as a readout for anaphase-promoting complex/cyclosome activity, we find that suppression of kinetochore stretching delays and decelerates cyclin B degradation. These observations suggest that the SAC monitors stretching of kinetochores rather than centromeres and that kinetochore stretching promotes silencing of the SAC signal.     

In Vivo Messenger RNA Introduction into the Central Nervous System Using Polyplex Nanomicelle

Messenger RNA (mRNA) introduction is a promising approach to produce therapeutic proteins and peptides without any risk of insertion mutagenesis into the host genome. However, it is difficult to introduce mRNA in vivo mainly because of the instability of mRNA under physiological conditions and its strong immunogenicity through the recognition by Toll-like receptors (TLRs). We used a novel carrier based on self-assembly of a polyethylene glycol (PEG)-polyamino acid block copolymer, polyplex nanomicelle, to administer mRNA into the central nervous system (CNS). The nanomicelle with 50 nm in diameter has a core-shell structure with mRNA-containing inner core surrounded by PEG layer, providing the high stability and stealth property to the nanomicelle. The functional polyamino acids possessing the capacity of pH-responsive membrane destabilization allows smooth endosomal escape of the nanomicelle into the cytoplasm. After introduction into CNS, the nanomicelle successfully provided the sustained protein expression in the cerebrospinal fluid for almost a week. Immune responses after mRNA administration into CNS were effectively suppressed by the use of the nanomicelle compared with naked mRNA introduction. In vitro analyses using specific TLR-expressing HEK293 cells confirmed that the nanomicelle inclusion prevented mRNA from the recognition by TLRs. Thus, the polyplex nanomicelle is a promising system that simultaneously resolved the two major problems of in vivo mRNA introduction, the instability and immunogenicity, opening the door to various new therapeutic strategies using mRNA.     

Ig L-chain shuffling for affinity maturation of phage library-derived human anti-human MCP-1 antibody blocking its chemotactic activity

Monocyte chemotactic protein-1 (MCP-1, CC-chemokine ligand 2; CCL2) is involved in the development of various forms of chronic inflammations. Employing the naive human single-chain Fv displaying phage library, we established seven MCP-1-specific scFvs. The MC8 and MC32 clones exhibited blocking activity for the MCP-1-induced chemotaxis of THP-1 cells, in spite of their monovalency. The analysis of V gene usage showed that all clones bore the identical Vh1 gene, IGHV1-24*01, with variable DJ joining sequences, while their Vl usage was relatively varied, suggesting the preferential contribution of the Vh gene. Based on these findings, to minimize the deteriorative influences on the MCP-1 specificity of MC32, we aimed to achieve the affinity maturation of MC32 using MC32 L-chain shuffling library and select MC32 variants. Most MC32 variants increased their affinity by reducing the k(off) value with no influence of the antigen specificity. MC32 variants #22 or #56 showed approximately 15-fold higher affinity than MC32, indicating that the L-chain shuffling library is useful if the Vh is dominantly involved in the determination of the antigen specificity.