Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ

BACKGROUND: Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. METHODOLOGY/PRINCIPAL FINDINGS: To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids. CONCLUSIONS/SIGNIFICANCE: From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori

Laparoscopic Findings of Rare Pediatric Inguinal Hernias

Pediatric inguinal hernias are caused to the patency of the processus vaginalis (PPV). The principle for the repair of indirect inguinal hernias in children consists of complete ligation of the PPV. Laparoscopic percutaneous extraperitoneal closure (LPEC) has spread rapidly since it was reported by some groups from around 1998, and the number of institutions adopting this method as a standard procedure for pediatric inguinal hernia is increasing in Japan. Since the closure of PPV by laparoscopic surgery is popular, rare hernias in children can be observed from the abdominal cavity. We present the laparoscopic findings of rare pediatric inguinal hernias and report their experience

Enzymatic control of anhydrobiosis-related accumulation of trehalose in the sleeping chironomid, Polypedilum vanderplanki

Larvae of an anhydrobiotic insect, Polypedilum vanderplanki, accumulate very large amounts of trehalose as a compatible solute on desiccation, but the molecular mechanisms underlying this accumulation are unclear. We therefore isolated the genes coding for trehalose metabolism enzymes, i.e. trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) for the synthesis step, and trehalase (TREH) for the degradation step. Although computational prediction indicated that the alternative splicing variants (PvTpsα/β) obtained encoded probable functional motifs consisting of a typical consensus domain of TPS and a conserved sequence of TPP, PvTpsα did not exert activity as TPP, but only as TPS. Instead, a distinct gene (PvTpp) obtained expressed TPP activity. Previous reports have suggested that insect TPS is, exceptionally, a bifunctional enzyme governing both TPS and TPP. In this article, we propose that TPS and TPP activities in insects can be attributed to discrete genes. The translated product of the TREH ortholog (PvTreh) certainly degraded trehalose to glucose. Trehalose was synthesized abundantly, consistent with increased activities of TPS and TPP and suppressed TREH activity. These results show that trehalose accumulation observed during anhydrobiosis induction in desiccating larvae can be attributed to the activation of the trehalose synthetic pathway and to the depression of trehalose hydrolysis

One-Step Detection of the 2009 Pandemic Influenza A(H1N1) Virus by the RT-SmartAmp Assay and Its Clinical Validation

<div><h3>Background</h3><p>In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society.</p> <h3>Methodology</h3><p>To address the clinical need for rapid diagnosis, we have developed a new method, the “RT-SmartAmp assay”, to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses.</p> <h3>Results and Conclusions</h3><p>We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.</p> </div

クロレラからの血圧降下作用糖蛋白質の検索(農芸化学部門)

クロレラから, 血圧降下作用物質が部分精製された。生クロレラの細胞壁破砕物を冷アセトンにて脱脂脱色後, 冷水抽出物が得られた。冷水抽出物の80%エタノール不溶性画分に降圧作用が認められた。80%エタノール不溶性画分のBio-Gel P-60ゲルロ過, DEAE-セルロースクロマトグラフィーにより, ほぼ均一な降圧画分D-IIが得られた。画分D-IIの分子量は, 約80,000と推定された。画分D-IIを, 13週令の血圧180&acd;190mmHgのSHRに0.5mg/100g体重の割合で腹腔内投与すると, 1時間後に140&acd;160mmHgという血圧になり, 強い降圧効果が認められた。画分D-IIは, ガラクトース, マンノース, グルコサミンを含む糖蛋白質で, アスパラギン酸, グルタミン酸, アラニン, プロリンの含量が高く, 塩基性アミノ酸が少なかった。The antihypertensive material was partially purified from chlorella. Cell wall of chlorella was destroyed with Dyno-mill. After defatted and decolored with cold aceton, water extract was obtained from chlorella by the extraction at 4℃ for 24 hours. 80% ethanol precipitate from water extract was fractionated by Bio-Gel P-60 gel filtration followed by DEAE-cellulose chromatatography. About 50mg of antihypertensive fraction D-II were obtained from 10g of chlorella. The molecular weight of fraction D-II was about 80,000 dalton. The blood pressure of spontaneously hypertensive rat (13 weeks of age) was decreased from 185 to 150mmHg, when fraction D-II was injected intraperitoneally at the concentration of 0.5mg/100g of body weight. Fraction D-II contained galactose, mannose and glucosamine. The contents of aspartic acid, glutamic acid, alanine and proline in fraction D-II were large

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution

Conditional Rather than Absolute Requirements of the Capsid Coding Sequence for Initiation of Methionine-Independent Translation in Plautia stali Intestine Virus

The positive-stranded RNA genome of Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) in an intergenic region (IGR). The IGR-IRES of PSIV initiates translation of the capsid protein by using CAA, the codon for glutamine. It was previously reported (J. Sasaki and N. Nakashima, J. Virol. 73:1219-1226, 1999) that IGR-IRES extended by several nucleotides into the capsid open reading frame (ORF). Despite the fact that the secondary structure model of the IGR-IRES is highly conserved, we were unable to find structural similarities in the 5′ region of the capsid ORFs in related viruses. Therefore, we reevaluated the role of the capsid ORF in IGR-IRES-mediated translation in PSIV. Mutation of the CAA codon with various triplets did not inhibit IGR-IRES-mediated translation. N-terminal amino acid analyses of mutated products showed that the IGR-IRES could initiate translation by using various elongator tRNAs. By replacement of the capsid ORF with exogenous coding sequences having AUG deleted, translation products were produced in most cases, but capsid-exogenous fusion proteins were produced more efficiently than were the translation products. These data indicate that the 5′ part of the capsid ORF is not an absolute requirement for the IGR-IRES-mediated translation. RNA structure probing analyses showed that the 5′ part of the capsid ORF was a single strand, while that of exogenous reading frames was structured. Exogenous sequences also caused structural distortion in the 3′ part of the IGR-IRES. We hypothesize that the single-stranded capsid ORF helps to form the tertiary structure of the IGR-IRES and facilitates precise positioning of ribosomes

Conserved Protein Kinases Encoded by Herpesviruses and Cellular Protein Kinase cdc2 Target the Same Phosphorylation Site in Eukaryotic Elongation Factor 1δ

Earlier studies have shown that translation elongation factor 1δ (EF-1δ) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1δ associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1δ by alanine precluded the posttranslational processing of EF-1δ, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1δ containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1δ completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1δ. (v) As observed with EF-1δ, the casein kinase II β subunit (CKIIβ) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIβ by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells

Primary great saphenous vein aneurysm in a five-year-old boy

AbstractPrimary venous aneurysm is a very rare disease, especially in pediatric patients. A case of primary great saphenous vein aneurysm in a five-year-old boy is reported. He was initially suspected of suffering from inguinal hernia because the soft mass was detected at the inguinal region when the patient was in the standing position. However, ultrasonography revealed the swelling to be a great saphenous vein aneurysm. The lesion was restricted to the thigh area and was surgically excised