Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system

Six types of recombination signal DNA sequences of the Multisite Gateway cloning system were investigated as to their specificity and efficiency in the LR and BP recombination reactions. In the LR reaction to generate an Expression clone by recombination between attL and attR signals which are contained in the Entry clone and the Destination vector, respectively, the cross-reactivity of various attL and attR pairs on six types of respective signal sequences was examined. In the BP reaction to create an Entry clone by transferring the target DNA segment in the Expression clone or the attB-flanked PCR product into a Donor vector, various combinations of attB and attP pairs were tested for their reactivities in recombination. The results obtained indicate a markedly higher specificity and efficiency of cross-reactivity with only the matched att signal pairs, such as attL3-attR3, attB5-attP5, and so on, compared to unmatched signal pairs, such as attL3-attR5, attB5-attP3, and so on, thus verifying a high-throughput production of the positive clones in the Gateway system in which multiple recombination signals exist together in one reaction system. Examples of rapid construction of a three or four DNA-fusion structure in the plasmid are shown.     

The Plasmodium knowlesi MAHRP2 ortholog localizes to structures connecting Sinton Mulligan's clefts in the infected erythrocyte

During development within the host erythrocyte malaria parasites generate nascent membranous structures which serve as a pathway for parasite protein transport to modify the host cell. The molecular basis of such membranous structures is not well understood, particularly for malaria parasites other than Plasmodium falciparum. To characterize the structural basis of protein trafficking in the Plasmodium knowlesi-infected erythrocyte, we identified a P. knowlesi ortholog of MAHRP2, a marker of the tether structure that connects membranous structures in the P. falciparum-infected erythrocyte. We show that PkMAHRP2 localizes on amorphous structures that connect Sinton Mulligan's clefts (SMC) to each other and to the erythrocyte membrane. Three dimensional reconstruction of the P. knowlesi-infected erythrocyte revealed that the SMC is a plate-like structure with swollen ends, reminiscent of the morphology of the Golgi apparatus. The PkMAHRP2-localized amorphous structures are possibly functionally equivalent to P. falciparum tether structure. These findings suggest a conservation in the ultrastructure of protein trafficking between P. falciparum and P. knowlesi.     

Functional human T lymphocyte development from cord blood CD34+ cells in nonobese diabetic/Shi-scid,IL-2 receptor gamma null mice

An experimental model for human T lymphocyte development from hemopoietic stem cells is necessary to study the complex processes of T cell differentiation in vivo. In this study, we report a newly developed nonobese diabetic (NOD)/Shi-scid, IL-2Rgamma null (NOD/SCID/gamma(c)(null)) mouse model for human T lymphopoiesis. When these mice were transplanted with human cord blood CD34(+) cells, the mice reproductively developed human T cells in their thymus and migrated into peripheral lymphoid organs. Furthermore, these T cells bear polyclonal TCR-alphabeta, and respond not only to mitogenic stimuli, such as PHA and IL-2, but to allogenic human cells. These results indicate that functional human T lymphocytes can be reconstituted from CD34(+) cells in NOD/SCID/gamma(c)(null) mice. This newly developed mouse model is expected to become a useful tool for the analysis of human T lymphopoiesis and immune response, and an animal model for studying T lymphotropic viral infections, such as HIV.     

Stable allele frequency distribution of the polymorphic region of SURFIN(4.2) in Plasmodium falciparum isolates from Thailand

Plasmodium falciparum SURFIN_4.2 (PFD1160w) is a polymorphic protein expressed on the surface of parasite-infected erythrocytes. Such molecules are expected to be under strong host immune pressure, thus we analyzed the nucleotide diversity of the N-terminal extracellular region of SURFIN_4.2 using P. falciparum isolates obtained from a malaria hypoendemic area of Thailand. The extracellular region of SURFIN_4.2 was divided into four regions based on the amino acid sequence conservation among SURFIN members and the level of polymorphism among SURFIN_4.2 sequences; N-terminal segment (Nter), a cysteine-rich domain (CRD), a variable region 1 (Var1), and a variable region 2 (Var2). Comparison between synonymous and non-synonymous substitutions, Tajima's D test, and Fu and Li's D* and F* tests detected signatures of positive selection on Var2 and to a lesser extent Var1, suggesting that these regions were likely under host immune pressure. Strong linkage disequilibrium was detected for nucleotide pairs separated by a distance of more than 1.5 kb, and 7 alleles among 19 alleles detected in 1988–1989 still circulated 14 years later, suggesting low recombination of the analyzed surf_4.2 sequence region in Thailand. The allele frequency distribution of polymorphic areas in Var2 did not differ between two groups collected in different time points, suggesting the allele frequency distribution of this region was stable for 14 years. The observed allele frequency distribution of SURFIN_4.2 Var2 may be fixed in Thai P. falciparum population as similar to the observation for P. falciparum merozoite surface protein 1, for which a stable allele frequency distribution was reported.     

The N-terminal segment of Plasmodium falciparum SURFIN4.1 is required for its trafficking to the red blood cell cytosol through the endoplasmic reticulum

Plasmodium falciparum SURFIN is a type I transmembrane protein that shares domains with molecules expressed on the surface of the red blood cells (RBCs) infected with a variety of malaria parasite species, such as P. falciparum PfEMP1, Plasmodium vivax VIR proteins, and Plasmodium knowlesi SICAvar. Thus, understanding the export mechanism of SURFIN to the RBC may provide fundamental insights into how malaria parasites export their proteins to RBC cytosol in general. We re-evaluate SURFIN_4.1 for its exon–intron boundaries, location, and the function of each region by expressing recombinant SURFIN_4.1 in P. falciparum. We found that, in two 3D7 lines and one Thai isolate, SURFIN_4.1 possesses only 19 amino acids after the predicted transmembrane region, whereas in the FCR3 line, it possesses two tryptophan-rich domains in its intracellular region. Recombinant SURFIN_4.1 based on the 3D7 sequence was detected in the Maurer's clefts of infected RBCs, suggesting that endogenous SURFIN_4.1 is also exported to Maurer's clefts. Brefeldin A-sensitive export of SURFIN_4.1 indicates that its export is endoplasmic reticulum (ER)/Golgi-dependent. By sequential deletion and replacement with unrelated protein sequences, we find that the SURFIN_4.1 transmembrane region is essential for the initial recruitment of the protein to the ER, and the following sorting step to the parasitophorous vacuole is determined by two independent signals located in the N-terminus 50 amino acids. TM region with the adjacent cytoplasmic region also contains information for the efficient recruitment to the ER and/or for the efficient translocation across the parasitophorous vacuole membrane. We also found that SURFIN_4.1 might form a homomeric complex during the trafficking using cysteine rich domain and/or variable region.     

The upstream sequence segment of the C-terminal cysteine-rich domain is required for microneme trafficking of Plasmodium falciparum erythrocyte binding antigen 175

Erythrocyte invasion is a critical step for survival of Plasmodium parasites, the causative agents of malaria, in their host and recognition of the host cell receptors by Plasmodium erythrocyte-binding-like (EBL) proteins plays an important role. Although EBL subcellular localization was shown to be closely linked to parasite virulence in the rodent model of malaria, the trafficking of EBL to micronemes, the secretory organelle in the invasive parasite is not fully understood. In this study, we assessed the impact of the deletion and amino acid replacement of Plasmodium falciparum EBL (EBA-175) using transgenic P. falciparum lines expressing modified EBA-175. We found that, in addition to a signal peptide and a cysteine rich region (region 6) to the cytoplasmic tail, a previously unrecognized sequence segment in region 5 was required for correct microneme trafficking of EBA-175. Replacement of Arg or Phe residues in this segment altered microneme trafficking, suggesting that the sequence itself contained critical information. Based on these findings, we propose that the sequence segment in region 5 is also required for the recognition of EBA-175 by the trafficking machinery to direct this protein to the microneme. Our results provide key information to clarify an as yet unidentified EBA-175 trafficking mechanism.     

Modeling Alzheimer’s Disease with iPSCs Reveals Stress Phenotypes Associated with Intracellular Aβ and Differential Drug Responsiveness

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Highlights? Familial and sporadic Alzheimer’s patient iPSC-derived neural cells were analyzed ? Intracellular Aβ oligomers accumulate in lines from some patients ? Aβ oligomer accumulation is associated with ER and oxidative stress ? DHA-alleviated ER and oxidative stresses improve cell viability