Insulin signaling is involved in hematopoietic regulation in an insect hematopoietic organ

Only a few extracellular hematopoietic factors have been identified in insects. We previously developed an in vitro culture system for the larval hematopoietic organ (HPO) of the silkworm Bombyx mori, and found that cell proliferation is linked to hemocyte discharge from the HPO. In this study, we tested hematopoietic activity of bombyxin, a peptide in the insulin family. When silkworm HPO was cultured with synthetic bombyxin-II, the number of discharged hemocytes increased in a dose-dependent manner, indicating that bombyxin promoted cell proliferation in the HPO. However, a neutralization experiment using anti-bombyxin-II antibody revealed that bombyxin is not the primary effector in larval plasma. Similarly, bovine insulin showed hematopoietic activity. Addition of molting hormone, 20-hydroxyecdysone, circumstantially enhanced the hematopoietic activity of bombyxin and insulin. Bombyxin and insulin induced phosphorylation of different sets of proteins in the HPO, suggesting that their signaling pathways are different.     

Novel carbohydrate-binding activity of bovine liver beta-glucuronidase toward lactose/N-acetyllactosamine sequences

Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed.     

Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information

DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids. Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis, respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level. Chromosome mapping of these markers using a Lolium/Festuca substitution line revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of Anhydrobiosis-related Genes from an Expressed Sequence Tag Database in the Cryptobiotic Midge Polypedilum vanderplanki (Diptera; Chironomidae)

Some organisms are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. The sleeping chironomid (Polypedilum vanderplanki) lives in the semi-arid regions of Africa, and its larvae can survive desiccation in an anhydrobiotic form during the dry season. To unveil the molecular mechanisms of this resistance to desiccation, an anhydrobiosis-related Expressed Sequence Tag (EST) database was obtained from the sequences of three cDNA libraries constructed from P. vanderplanki larvae after 0, 12, and 36 h of desiccation. The database contained 15,056 ESTs distributed into 4,807 UniGene clusters. ESTs were classified according to gene ontology categories, and putative expression patterns were deduced for all clusters on the basis of the number of clones in each library; expression patterns were confirmed by real-time PCR for selected genes. Among up-regulated genes, antioxidants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (Hsps) were identified as important groups for anhydrobiosis. Genes related to trehalose metabolism and various transporters were also strongly induced by desiccation. Those results suggest that the oxidative stress response plays a central role in successful anhydrobiosis. Similarly, protein denaturation and aggregation may be prevented by marked up-regulation of Hsps and the anhydrobiosis-specific LEA proteins. A third major feature is the predicted increase in trehalose synthesis and in the expression of various transporter proteins allowing the distribution of trehalose and other solutes to all tissues.     

Mitochondrial protein import. Requirement of presequence elements and tom components for precursor binding to the TOM complex

Protein translocation across the outer mitochondrial membrane is mediated by the translocator called the TOM (translocase of the outer mitochondrial membrane) complex. The TOM complex possesses two presequence binding sites on the cytosolic side (the cis site) and on the intermembrane space side (the trans site). Here we analyzed the requirement of presequence elements and subunits of the TOM complex for presequence binding to the cis and trans sites of the TOM complex. The N-terminal 14 residues of the presequence of subunit 9 of F(0)-ATPase are required for binding to the trans site. The interaction between the presequence and the cis site is not sufficient to anchor the precursor protein to the TOM complex. Tom7 constitutes or is close to the trans site and has overlapping functions with the C-terminal intermembrane space domain of Tom22 in the mitochondrial protein import.     

Thyroid hormone regulates mRNA expression and currents of ion channels in rat atrium

Atrial fibrillation is one of the common arrhythmias associated with hyperthyroidism. This study examined the effects of thyroid hormone (T3) on mRNA expression and currents of major ionic channels determining the action potential duration (APD) in the rat atrium using the RNase protection assay and the whole-cell patch-clamp technique, respectively. T3 increased the Kv1.5 mRNA expression and decreased the L-type calcium channel mRNA expression, while the Kv4.2 mRNA expression did not change. APD was shorter in hyperthyroid than in euthyroid myocytes. The ultrarapid delayed rectifier potassium currents were remarkably increased in hyperthyroid than in euthyroid myocytes, whereas the transient outward potassium currents were unchanged. L-type calcium currents were decreased in hyperthyroid than in euthyroid myocytes. T3 shifted the current-voltage relationship for calcium currents negatively. In conclusion, T3 increased the outward currents and decreased the inward currents. The resultant changes of ionic currents shortened APD, providing a substrate for atrial fibrillation.     

Induction of C-reactive protein, serum amyloid P component, and kininogens in the submandibular and lacrimal glands of rats with experimentally induced inflammation.

The mRNAs for acute-phase proteins and kininogens were found to be increased in the submandibular gland (SMG) and extraorbital and intraorbital lacrimal gland (ELG and ILG) in response to experimentally induced inflammation in rats; i.e., 24 hours after subcutaneous injection of turpentine oil, mRNAs for C-reactive protein (CRP), serum amyloid P component (SAP), and H- and T-kininogens were induced in the SMG, ELG, and ILG of rats, whereas these mRNAs were not detected in the same tissues of normal control rats. The induction of mRNAs for these inflammatory proteins by turpentine oil was preceded by a transient increase in the level of mRNA for tumor necrosis factor-alpha (TNF-alpha) at 6 hours after subcutaneous injection of the oil. This was confirmed by injection of another inflammation inducer, lipopolysaccharide (LPS), which induced the TNF-alpha mRNA in the same way at 6 hours as turpentine oil did. The up-regulation of acute-phase proteins including kininogens in the SMG, ELG, and ILG suggest the existence of a strict defense system in the exocrine glands.