The 3′-Phosphoadenosine 5′-Phosphosulfate Transporters,PAPST1 and 2, Contribute to the Maintenance and Differentiation of Mouse Embryonic Stem Cells

Recently, we have identified two 3′-phosphoadenosine 5′-phosphosulfate (PAPS) transporters (PAPST1 and PAPST2), which contribute to PAPS transport into the Golgi, in both human and Drosophila. Mutation and RNA interference (RNAi) of the Drosophila PAPST have shown the importance of PAPST-dependent sulfation of carbohydrates and proteins during development. However, the functional roles of PAPST in mammals are largely unknown. Here, we investigated whether PAPST-dependent sulfation is involved in regulating signaling pathways required for the maintenance of mouse embryonic stem cells (mESCs), differentiation into the three germ layers, and neurogenesis. By using a yeast expression system, mouse PAPST1 and PAPST2 proteins were shown to have PAPS transport activity with an apparent K_m value of 1.54 µM or 1.49 µM, respectively. RNAi-mediated knockdown of each PAPST induced the reduction of chondroitin sulfate (CS) chain sulfation as well as heparan sulfate (HS) chain sulfation, and inhibited mESC self-renewal due to defects in several signaling pathways. However, we suggest that these effects were due to reduced HS, not CS, chain sulfation, because knockdown of mouse N-deacetylase/N-sulfotransferase, which catalyzes the first step of HS sulfation, in mESCs gave similar results to those observed in PAPST-knockdown mESCs, but depletion of CS chains did not. On the other hand, during embryoid body formation, PAPST-knockdown mESCs exhibited abnormal differentiation, in particular neurogenesis was promoted, presumably due to the observed defects in BMP, FGF and Wnt signaling. The latter were reduced as a result of the reduction in both HS and CS chain sulfation. We propose that PAPST-dependent sulfation of HS or CS chains, which is regulated developmentally, regulates the extrinsic signaling required for the maintenance and normal differentiation of mESCs.     

ECG Response of Koalas to Tourists Proximity: A Preliminary Study

Koalas operate on a tight energy budget and, thus, may not always display behavioral avoidance reaction when placed in a stressful condition. We investigated the physiological response of captive koalas Phascolarctos cinereus in a conservation centre to the presence of tourists walking through their habitat. We compared, using animal-attached data-recorders, the electrocardiogram activity of female koalas in contact with tourists and in a human-free area. One of the koalas in the tourist zone presented elevated heart rate values and variability throughout the recording period. The remaining female in the exhibit area showed a higher field resting heart rates during the daytime than that in the isolated area. In the evening, heart rate profiles changed drastically and both the koalas in the exhibit and in the tourist-free zones displayed similar field resting heart rates, which were lower than those during the day. In parallel, the autonomic nervous systems of these two individuals evolved from sympathetic-dominant during the day to parasympathetic-dominant in the evening. Our results report ECG of free-living koalas for the first time. Although they are preliminary due to the difficulty of having sufficient samples of animals of the same sex and age, our results stress out the importance of studies investigating the physiological reaction of animals to tourists.     

Lysosomal-Associated Protein Multispanning Transmembrane 5 Gene (LAPTM5) Is Associated with Spontaneous Regression of Neuroblastomas

Background

Neuroblastoma (NB) is the most frequently occurring solid tumor in children, and shows heterogeneous clinical behavior. Favorable tumors, which are usually detected by mass screening based on increased levels of catecholamines in urine, regress spontaneously via programmed cell death (PCD) or mature through differentiation into benign ganglioneuroma (GN). In contrast, advanced-type NB tumors often grow aggressively, despite intensive chemotherapy. Understanding the molecular mechanisms of PCD during spontaneous regression in favorable NB tumors, as well as identifying genes with a pro-death role, is a matter of urgency for developing novel approaches to the treatment of advanced-type NB tumors.

Principal Findings

We found that the expression of lysosomal associated protein multispanning transmembrane 5 (LAPTM5) was usually down-regulated due to DNA methylation in an NB cell-specific manner, but up-regulated in degenerating NB cells within locally regressing areas of favorable tumors detected by mass-screening. Experiments in vitro showed that not only a restoration of its expression but also the accumulation of LAPTM5 protein, was required to induce non-apoptotic cell death with autophagic vacuoles and lysosomal destabilization with lysosomal-membrane permeabilization (LMP) in a caspase-independent manner. While autophagy is a membrane-trafficking pathway to degrade the proteins in lysosomes, the LAPTM5-mediated lysosomal destabilization with LMP leads to an interruption of autophagic flux, resulting in the accumulation of immature autophagic vacuoles, p62/SQSTM1, and ubiqitinated proteins as substrates of autophagic degradation. In addition, ubiquitin-positive inclusion bodies appeared in degenerating NB cells.

Conclusions

We propose a novel molecular mechanism for PCD with the accumulation of autophagic vacuoles due to LAPTM5-mediated lysosomal destabilization. LAPTM5-induced cell death is lysosomal cell death with impaired autophagy, not cell death by autophagy, so-called autophagic cell death. Thus LAPTM5-mediated PCD is closely associated with the spontaneous regression of NBs and opens new avenues for exploring innovative clinical interventions for this tumor.     

Construction of Escherichia coli gene expression level perturbation collection

We generated 61 strains of Escherichia coli in which the expression level of a specific single gene can be changed continuously over a physiologically significant range. In each strain, one auxotrophic gene was deleted from its original position and reinserted at a specific position on the chromosome under the control of the tetA promoter. Therefore, the level of expression of the target gene can be controlled easily by altering the concentrations of inducers, e.g., anhydrotetracycline and doxycycline, in the medium. Protein and mRNA levels and changes in proliferation rate were examined in some of the strains in our collection to determine the ability to control the level of target gene expression over a physiologically significant range. These strains will be useful for extracting omics data sets and for the construction of genome-scale mathematical models, because causality between perturbations in gene expression level and their consequences can be clearly determined.     

Determination of pyrrole–imidazole polyamide in rat plasma by liquid chromatography–tandem mass spectrometry

Pyrrole (Py)–imidazole (Im) polyamides synthesized by combining N-methylpyrrole and N-methylimidazole amino acids have been identified as novel candidates for gene therapy. In this study, a sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with an electrospray ionization (ESI) source was developed and validated for the determination and quantification of Py–Im polyamide in rat plasma. Py–Im polyamide was extracted from rat plasma by solid-phase extraction (SPE) using a Waters Oasis^® HLB cartridge. Separation was achieved on an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 50 mm) column by gradient elution using acetonitrile:distilled water:acetic acid (5:95:0.1, v/v/v) and acetonitrile:distilled water:acetic acid (95:5:0.1, v/v/v). The method was validated over the range of 10–1000 ng/mL and the lower limit of quantification (LLOQ) was 10 ng/mL. This method was successfully applied to the investigation of the pharmacokinetics of Py–Im polyamide after intravenous administration.     

FON2 SPARE1 Redundantly Regulates Floral Meristem Maintenance with FLORAL ORGAN NUMBER2 in Rice

CLAVATA signaling restricts stem cell identity in the shoot apical meristem (SAM) in Arabidopsis thaliana. In rice (Oryza sativa), FLORAL ORGAN NUMBER2 (FON2), closely related to CLV3, is involved as a signaling molecule in a similar pathway to negatively regulate stem cell proliferation in the floral meristem (FM). Here we show that the FON2 SPARE1 (FOS1) gene encoding a CLE protein functions along with FON2 in maintenance of the FM. In addition, FOS1 appears to be involved in maintenance of the SAM in the vegetative phase, because constitutive expression of FOS1 caused termination of the vegetative SAM. Genetic analysis revealed that FOS1 does not need FON1, the putative receptor of FON2, for its action, suggesting that FOS1 and FON2 may function in meristem maintenance as signaling molecules in independent pathways. Initially, we identified FOS1 as a suppressor that originates from O. sativa indica and suppresses the fon2 mutation in O. sativa japonica. FOS1 function in japonica appears to be compromised by a functional nucleotide polymorphism (FNP) at the putative processing site of the signal peptide. Sequence comparison of FOS1 in about 150 domesticated rice and wild rice species indicates that this FNP is present only in japonica, suggesting that redundant regulation by FOS1 and FON2 is commonplace in species in the Oryza genus. Distribution of the FNP also suggests that this mutation may have occurred during the divergence of japonica from its wild ancestor. Stem cell maintenance may be regulated by at least three negative pathways in rice, and each pathway may contribute differently to this regulation depending on the type of the meristem. This situation contrasts with that in Arabidopsis, where CLV signaling is the major single pathway in all meristems.     

Asymmetric cell division of rice zygotes located in embryo sac and produced by in vitro fertilization

In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. This unequal division of the zygote is a putative first step for formation of the apical–basal axis of plants and is a fundamental feature of early embryogenesis and morphogenesis in angiosperms. Because fertilization and subsequent embryogenesis occur in embryo sacs, which are deeply embedded in ovular tissue, in vitro fertilization of isolated gametes is a powerful system to dissect mechanisms of fertilization and post-fertilization events. Rice is an emerging molecular and experimental model plant, however, profile of the first zygotic division within embryo sac and thus origin of apical–basal embryo polarity has not been closely investigated. Therefore, in the present study, the division pattern of rice zygote in planta was first determined accurately by observations employing serial sections of the egg apparatus, zygotes and two-celled embryos in the embryo sac. The rice zygote divides asymmetrically into a two-celled embryo consisting of a statistically significantly smaller apical cell with dense cytoplasm and a larger vacuolated basal cell. Moreover, detailed observations of division profiles of zygotes prepared by in vitro fertilization indicate that the zygote also divides into an asymmetric two-celled embryo as in planta. Such observations suggest that in vitro-produced rice zygotes and two-celled embryos may be useful as experimental models for further investigations into the mechanism and control of asymmetric division of plant zygotes.     

Cytoplasmic expression of the JM403 antigen GlcA-GlcNH 3 + on heparan sulfate glycosaminoglycan in mammary carcinomas—a novel proliferative biomarker for breast cancers with high malignancy

The expressions of heparan sulfate glycosaminoglycans (HSGAGs) in breast carcinoma specimens from 60 patients were immunohistochemically investigated using monoclonal antibodies (mAbs) that recognized different epitopes of the glycan structure. Cytoplasmic expression of GlcA-GlcNH ₃ ⁺ on HSGAG was detected in carcinomas at high frequency (58.3%) using mAb JM403, whereas it was almost undetectable in normal breast ducts. This cytoplasmic expression was confirmed using confocal laser scanning microscopy. The expression of JM403 antigen in invasive carcinomas significantly correlated with nuclear atypia score (p?=?0.0004), mitotic counts score (p?=?0.0018), nuclear grade (p?=?0.0061) and the incidence of metastasis to axillary lymph nodes (p?=?0.0061). Furthermore, its expression was significantly correlated with the Ki67-labeling index in 55 invasive carcinomas (p?p? ₃ ⁺ was also expressed in the cytoplasm of normal crypt epithelial cells where Ki67 protein was expressed in the cell nuclei in the proliferative compartment of the human small intestines. To date, HSGAGs have generally been found to exist on cell surface membranes and in extracellular matrices as components of HS proteoglycans, and the negatively-charged sulfated domains on HSGAGs are considered to be important for their functions. However, our present findings indicate that the cytoplasmic expression of the JM403 antigen GlcA-GlcNH ₃ ⁺ on positively charged, non-sulfated HSGAG may be involved in cell proliferation and associated with increased degrees of malignancy. The unordinary carbohydrate antigen of GlcA-GlcNH ₃ ⁺ on HSGAGs recognized by mAb JM403 may represent a novel proliferative biomarker for highly malignant mammary carcinomas.     

Codon reassignment in the Escherichia coli genetic code

Most organisms, from Escherichia coli to humans, use the ‘universal’ genetic code, which have been unchanged or ‘frozen’ for billions of years. It has been argued that codon reassignment causes mistranslation of genetic information, and must be lethal. In this study, we successfully reassigned the UAG triplet from a stop to a sense codon in the E. coli genome, by eliminating the UAG-recognizing release factor, an essential cellular component, from the bacterium. Only a few genetic modifications of E. coli were needed to circumvent the lethality of codon reassignment; erasing all UAG triplets from the genome was unnecessary. Thus, UAG was assigned unambiguously to a natural or non-natural amino acid, according to the specificity of the UAG-decoding tRNA. The result reveals the unexpected flexibility of the genetic code.     

Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.Transmembrane proteins play critical roles in cellular metabolism, participating in processes such as ion transport, nutrient uptake, signal transduction, and intercellular communication. As evidence of the essential functions of these proteins, more than half of all drug targets have been shown to be transmembrane proteins, and the analysis of the interactions of transmembrane proteins and their ligands is one of the most promising avenues for the discovery of new drug candidates. As a means of producing sufficient amounts of transmembrane proteins for binding analyses, heterologous protein expression systems have been developed using Escherichia coli (10), yeast (16), insect, and mammalian (4) cells as hosts. Transmembrane proteins generally are expressed at low levels and are extremely hydrophobic, rendering the analysis of interactions with ligands very difficult. In all cases, the analysis of membrane proteins requires a lipid or similar synthetic environment to maintain the native structure and function of the proteins. The purification of transmembrane proteins from cells frequently is time-consuming and typically results in the loss of the proteins’ native conformation.Magnetospirillum magneticum AMB-1 synthesizes intracellular nanosized bacterial magnetic particles (BacMPs; 50 to 100 nm); these are surrounded by a lipid bilayer membrane and exhibit strong ferrimagnetism. Functional soluble proteins have been expressed on BacMP surfaces through gene fusion techniques (11, 21, 24, 27) using BacMP membrane proteins (MagA, Mms16, and Mms13) as anchor proteins; this approach permits heterologous proteins to be localized efficiently and oriented appropriately on BacMPs. In a previous report, we demonstrated the successful display of the D1 dopamine receptor, a G protein-coupled receptor possessing seven transmembrane domains, on BacMPs. Mms16-D1, an dopamine receptor fusion protein, was expressed under the mms16 promoter, and a ligand-binding assay was performed (28). The assembly of transmembrane proteins on magnetic particles provides significant advantages for binding assays, including the easing of the purification of target proteins from bacterial cells without the loss of native conformation and the availability of a fully automated bioassay using robotic magnetic separation. Despite these advantages, there are not enough studies for the overexpression of transmembrane proteins other than the D1 dopamine receptor in M. magneticum AMB-1 because of its difficulty. In other host cells, a system for controlling gene expression has been employed to overcome its difficulty, and some successful efforts had achieved this for crystal structure analysis (5, 15, 18). The lack of these systems for M. magneticum has hampered the extension of this application to other transmembrane proteins.In this study, the tetracycline-inducible expression system was adapted for displaying transmembrane proteins on BacMPs in M. magneticum AMB-1. Expression vectors carrying the tetracycline repressor gene (tetR) and the target gene under the control of a strong promoter and the tetracycline operator (tetO) sequence were constructed, and the function of the system was evaluated using reporter genes. Finally, this system was applied to the overexpression of the transmembrane protein, tetraspanin CD81. This is the first report of an inducible expression system in M. magneticum, and it the demonstrates efficient display of a transmembrane protein at the surface of BacMPs.