HMGA1a is involved in specific splice site regulation of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) utilizes a highly complex splice site regulation system, taking advantage of host proteins, to express its own viral protein in an orderly way. We show here that one of the host proteins, high mobility group A protein 1a (HMGA1a), is involved in splice site regulation of 3′ splice site 2 (A2) and 5′splice site 3 (D3) of HIV-1 genomic RNA. shRNA knockdown of HMGA1 in HeLa cells resulting in a decrease of HMGA1 showed a significant decrease of Vpr mRNA. RNA electophoretic mobility shift assays showed HMGA1a specifically binds to a sequence adjacently upstream D3. In vitro splicing using heterologous pre-mRNA with A2 and D3, showed HMGA1a induced a splicing intermediate which decreased when an RNA decoy of the HMGA1a binding site was added. RT-PCR of in vitro splicing products revealed that HMGA1a induced an incomplete splicing product resulting from usage of A2 but inhibition of D3, which is reminiscent of the splicing pattern necessary for Vpr mRNA formation. HMGA1a interacted with hnRNPA1 shown by coimmunoprecipitation and supershifted U1 snRNP in an RNA electophoretic mobility shift assay. We conclude that HMGA1a anchors U1 snRNP to inhibit D3 function, and that HMGA1a inhibits hnRNPA1 function on exon splicing silencer of Vpr (ESSV) to activate A2 function. We show here for the first time that HMGA1a is involved in specific splice site regulation of HIV-1.     

Identification of Inter-Organ Vascular Network: Vessels Bridging between Organs

Development and homeostasis of organs and whole body is critically dependent on the circulatory system. In particular, the circulatory system, the railways shuttling oxygen and nutrients among various organs, is indispensible for inter-organ humoral communication. Since the modern view of the anatomy and mechanics of the circulatory system was established in 17^th century, it has been assumed that humoral factors are carried to and from organs via vascular branches of the central arteries and veins running along the body axis. Over the past few decades, major advances have been made in understanding molecular and cellular mechanisms underlying the vascularization of organs. However, very little is known about how each organ is linked by vasculature (i.e., inter-organ vascular networks). In fact, the exact anatomy of inter-organ vascular networks has remained obscure. Herein, we report the identification of four distinct vessels, V1^LP, V2^LP, V3^LP and V4^LP, that bridge between two organs, liver and pancreas in developing zebrafish. We found that these inter-organ vessels can be classified into two types: direct and indirect types. The direct type vessels are those that bridge between two organs via single distinct vessel, to which V1^LP and V2^LP vessels belong. The indirect type bridges between two organs via separate branches that emanate from a stem vessel, and V3^LP and V4^LP vessels belong to this type. Our finding of V1^LP, V2^LP, V3^LP and V4^LP vessels provides the proof of the existence of inter-organ vascular networks. These and other yet-to-be-discovered inter-organ vascular networks may facilitate the direct exchange of humoral factors that are necessary for the coordinated growth, differentiation and homeostasis of the connected organs. It is also possible that the inter-organ vessels serve as tracks for their connected organs to follow during their growth to establish their relative positions and size differences.     

Isoquinoline derivatives as potent CRTH2 antagonists: Design,synthesis and SAR

In this study, we describe the synthesis and structure–activity relationship (SAR) of a series of isoquinoline chemoattractant receptor–homologous molecule expressed on Th2 cells (CRTH2) antagonists. TASP0376377 (15-20), one of the most potent compounds, showed a potent binding affinity (IC₅₀ = 19 nM) in addition to the excellent functional antagonist activity (IC₅₀ = 13 nM). Moreover, the efficacy of this compound in a chemotaxis assay (IC₅₀ = 23 nM) was in good agreement with its potency as a CRTH2 antagonist. In addition, 15-20 exhibited greater selectivity in binding to CRTH2 than to the DP1 prostanoid receptor (IC₅₀ >1 μM) or the enzymes COX-1 and COX-2 (IC₅₀ >10 μM).     

Bacillus thuringiensis Cry toxins bound specifically to various proteins via domain III, which had a galactose-binding domain-like fold

Cry toxins have been reported to bind not only to receptors on insect cells but also to several unrelated proteins. In this study, we investigated the binding properties of Bacillus thuringiensis Cry toxins, focusing on domain III, a Cry toxin region with a structure that of the galactose-binding domain-like. Cry1Aa, Cry1Ac, and Cry8Ca specifically bound to several proteins unrelated to insect midgut cells. Cry1Aa binding to Cry toxin-binding proteins was inhibited by a monoclonal antibody, 2C2, indicating that Cry1Aa binds to these Cry toxin-binding proteins through domain III. Cry1Aa binding to Bombyx mori aminopeptidase N and other Cry toxin-binding proteins was inhibited by carbonic anhydrase, a Cry toxin-binding protein. The binding regions of carbonic anhydrase and Bombyx mori aminopeptidase N were narrowed to regions of less than 20 amino acids that did not have any similarity, suggesting that Cry toxin domain III has a binding pocket for multiple proteins.     

Identification of an estrogenic hormone receptor in Caenorhabditis elegans

Changes in both behavior and gene expression occur in Caenorhabditis elegans following exposure to sex hormones such as estrogen and progesterone, and to bisphenol A (BPA), an estrogenic endocrine-disrupting compound. However, only one steroid hormone receptor has been identified. Of the 284 known nuclear hormone receptors (NHRs) in C. elegans, we selected nhr-14, nhr-69, and nhr-121 for analysis as potential estrogenic hormone receptors, because they share sequence similarity with the human estrogen receptor. First, the genes were cloned and expressed in Escherichia coli, and then the affinity of each protein for estrogen was determined using a surface plasmon resonance (SPR) biosensor. All three NHRs bound estrogen in a dose-dependent fashion. To evaluate the specificity of the binding, we performed a solution competition assay using an SPR biosensor. According to our results, only NHR-14 was able to interact with estrogen. Therefore, we next examined whether nhr-14 regulates estrogen signaling in vivo. To investigate whether these interactions actually control the response of C. elegans to hormones, we investigated the expression of vitellogenin, an estrogen responsive gene, in an nhr-14 mutant. Semi-quantitative RT-PCR showed that vitellogenin expression was significantly reduced in the mutant. This suggests that NHR-14 is a C. elegans estrogenic hormone receptor and that it controls gene expression in response to estrogen.     

Bld10p constitutes the cartwheel-spoke tip and stabilizes the 9-fold symmetry of the centriole

Centrioles/basal bodies have a characteristic cylindrical structure consisting of nine triplet microtubules arranged in a rotational symmetry. How this elaborate structure is formed is a major unanswered question in cell biology [1, 2]. We previously identified a 170 kDa coiled-coil protein essential for the centriole formation in Chlamydomonas. This protein, Bld10p, is the first protein shown to localize to the cartwheel, a 9-fold symmetrical structure possibly functioning as the scaffold for the centriole-microtubule assembly [3]. Here, we report results by using a series of truncated Bld10p constructs introduced into a bld10 null mutant. Remarkably, a transformant (DeltaC2) in which 35% of Bld10p at the C terminus was deleted assembled centrioles with eight symmetrically arranged triplets, in addition to others with the normal nine triplets. The cartwheels in these eight-membered centrioles had spokes approximately 24% shorter than those in the wild-type, suggesting that the eight-triplet centrioles were formed because the cartwheel's smaller diameter. From the morphology of the cartwheel spoke in the DeltaC2 centriole and immunoelectron-microscope localization, we conclude that Bld10p is a major spoke-tip component that extends the cartwheel diameter and attaches triplet microtubules. These results provide the first experimental evidence for the crucial function of the cartwheel in centriolar assembly.     

Seasonal changes in taxon richness and abundance of mobile invertebrates inhabiting holdfast of annual kelp Ecklonia radicosa (Phaeophyceae,Lessoniaceae) at the central Pacific coast of Japan

To investigate seasonal changes in the taxon richness and abundance of mobile invertebrates inhabiting holdfasts of the warm temperate annual kelp Ecklonia radicosa, five holdfasts were collected monthly at the central Pacific coast of Japan from April to November 2014. During the study period, there was little variation in holdfast height and diameter, which ranged from 5.9 to 8.5 cm and from 7.1 to 10.8 cm, respectively. In total 7087 animals were collected from 40 holdfasts (177.2 individuals inds./holdfast, on average). The number of mobile invertebrates gradually increased from May (15 ± 9.9 inds./holdfast) to August (346 ± 152.5 inds./holdfast), with over 300 inds./holdfast until October before rapidly decreasing in November (110 ± 85.6 inds./holdfast). Similarly, taxon richness increased gradually from April (4.3 ± 1.0 taxa/holdfast) to August (11.0 ± 3.7 taxa/holdfast), and decreased in November (8.6 ± 2.3 taxa/holdfast). Interestingly, hundreds of mobile invertebrates inhabited holdfasts of kelp plants that had shed their blade in October and November. Taxon composition from August to October and the number of invertebrates from July to November were comparable to data previously reported for perennial kelps. In this study, the importance of annual Ecklonian species as biogenic habitats was demonstrated for the first time.     

DNA from pre-erythrocytic stage malaria parasites is detectable by PCR in the faeces and blood of hosts

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be “sub-microscopic” blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.