Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.     

New and highly efficient method for silkworm transgenesis using Autographa californica nucleopolyhedrovirus and piggyBac transposable elements

We have developed a new method for the transgenesis of the silkworm, Bombyx mori. This method couples the use of recombinant baculoviruses with the use of the piggyBac transposable element. One recombinant AcNPV, designated the helper virus, is designed to express the piggyBac transposase under the control of the Drosophila hsp70 promoter. Another recombinant AcNPV encoded the gene to be incorporated into the silkworm genome, in this case a green fluorescent protein (GFP) gene, under the control of B. mori actin A3 promoter and franked by the piggyBac inverted terminal repeats. Preblastoderm eggs were inoculated with a fine needle coated with a mixture of these two recombinant baculoviruses. Most of the inoculated larvae hatched and a high proportion of the newly hatched G0 larvae expressed the GFP marker. Transgenesis was confirmed by Southern blot analysis of G1 insects, sequencing the insertion site junctions isolated by inverse PCR, and the marker segregated in Mendelian fashion, as evidenced by the appearance of green fluorescence in G2 insects. Thus, transgenic silkworms were easily and efficiently obtained using this new method.     

Ultrastructural and immunochemical features of the cell wall sac formed in mulberry (Morus alba) idioblasts

A peculiar inward growth, named a “cell wall sac”, formed in mulberry (Morus alba) idioblasts, is a subcellular site for production of calcium carbonate crystals. On the basis of ultrastructural observations, a fully expanded cell wall sac could be divided into two parts—an amorphous complex consisting of multi-layered compartments with multiple fibers originating from the innermost cell wall layer, and a peripheral plain matrix with fiber aggregates. Immunofluorescent localization showed that low and highly esterified pectin epitopes were detected at the early stages of development of the cell wall sac, followed by complete disappearance from the both parts of fully enlarged mature sac. In contrast, the xyloglucan epitope remained in the compartment complex; this was supported by the observation that the xyloglucan epitope labeled with immuno-gold particles is found on fibers in the complex part.     

High Efficiency Selection of Full-length cDNA by Improved Biotinylated Cap Trapper

We report here an improved protocol for the preparation of full-lengthcDNA libraries that improves the previously reported method(Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics,137, 327–336), that allows long cDNAs to be cloned moreefficiently. One potential disadvantage of the original biotinylatedCAP trapper protocol is the exposure of mRNA to chemical andenzymatic attacks during the biotinylation of the cap structure,before the first-strand cDNA synthesis (and selection of full-lengthcDNA by biotinylated cap). Here, we show that the biotinylationof the cap structure is very specific and effective even ifbiotinylation is performed on the mRNA/cDNA hybrid producedby the first-strand cDNA synthesis reaction. Consequently, mRNAremains protected from chemical and enzymatic degradation duringthe overnight biotinylation step, thus making it possible toselect full-length cDNAs of longer average size. We herein reportthe efficiency and specificity of the new version of the protocolfor cap structure biotinylation and capture of full-length cDNA.     

Visual recognition of age class and preference for infantile features: implications for species-specific vs universal cognitive traits in primates

Despite not knowing the exact age of individuals, humans can estimate their rough age using age-related physical features. Nonhuman primates show some age-related physical features; however, the cognitive traits underlying their recognition of age class have not been revealed. Here, we tested the ability of two species of Old World monkey, Japanese macaques (JM) and Campbell's monkeys (CM), to spontaneously discriminate age classes using visual paired comparison (VPC) tasks based on the two distinct categories of infant and adult images. First, VPCs were conducted in JM subjects using conspecific JM stimuli. When analyzing the side of the first look, JM subjects significantly looked more often at novel images. Based on analyses of total looking durations, JM subjects looked at a novel infant image longer than they looked at a familiar adult image, suggesting the ability to spontaneously discriminate between the two age classes and a preference for infant over adult images. Next, VPCs were tested in CM subjects using heterospecific JM stimuli. CM subjects showed no difference in the side of their first look, but looked at infant JM images longer than they looked at adult images; the fact that CMs were totally na?ve to JMs suggested that the attractiveness of infant images transcends species differences. This is the first report of visual age class recognition and a preference for infant over adult images in nonhuman primates. Our results suggest not only species-specific processing for age class recognition but also the evolutionary origins of the instinctive human perception of baby cuteness schema, proposed by the ethologist Konrad Lorenz.     

Identification of the sperm-activating factor initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori

Male Bombyx mori has a trypsin-type protease, called initiatorin, in the secretion from the posterior segment of the ejaculatory duct that is thought to be involved in the acquisition of sperm motility, although this inference remains to be demonstrated. Here, we revised the experimental procedures including that for purification and definitely identified the purified initiatorin protein as an activation factor of B. mori sperm by an in vitro study in which we treated isolated spermatozoa with this enzyme. Analysis of cDNA revealed that initiatorin consists of 281 amino acids with sequence similarity to bovine trypsin, and is highly homologous to the ejaculated accessory gland proteins not only of other Lepidoptera but also of Orthoptera. Recombinant initiatorin, expressed in Escherichia coli and purified, also showed proteolytic and sperm-activating activities. RT-PCR and Western blot analyses indicated that initiatorin is abundantly expressed in the glandula (g.) prostatica. It was also shown that pro-initiatorin is synthesized and stored in g. prostatica, and then converted to the mature form upon ejaculation. Fluorogenic peptides with a dibasic sequence were efficiently cleaved by initiatorin, and one such substrate, BOC-Gly-Arg-Arg-MCA, inhibited sperm activation by the extract of g. prostatica. These results delineate the idea that initiatorin has the most suitable protease property as an initiator of the protein degradation cascade in that it releases free arginines, which in turn become an energy resource for sperm motility.     

Identification of two arginases generated by alternative splicing in the silkworm, Bombyx mori

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of arginine to ornithine and urea. Here, we have cloned two arginase cDNAs from the silkworm, Bombyx mori. The analysis of exon/intron structures showed that the two mRNAs named bmarg-r and bmarg-f were generated from a single gene by alternative usage of exons. The bmarg-r and bmarg-f were predicted to encode almost the same amino acid sequences, except that the latter had additional ten N-terminal residues. Recombinant bmARG-r and bmARG-f in Escherichia coli cell lysates were roughly similar to each other in enzymatic characteristics, which did not show large difference from those of arginases assayed by using tissue extracts. Differential RT-PCR experiments and tissue distribution analyses of arginase activity indicated that the bmarg-r gene is expressed in the male reproductive organs, especially in the glandula lacteola and vesicular seminalis, from which it is secreted to the seminal fluid and transferred to the female during copulation, whereas the bmarg-f gene is expressed in the larval and adult nonreproductive organs including the fat body and muscle, where the produced arginase proteins are considered to stay in the cells. Thus, the two silkworm arginase isoforms may have a difference in whether or not the product is excreted out of the cells in which it is synthesized.     

Impact of Age-Dependent Adventitia Inflammation on Structural Alteration of Abdominal Aorta in Hyperlipidemic Mice

Background

The adventitia is suggested to contribute to vascular remodeling; however, the site-selective inflammatory responses in association with the development of atherosclerosis remain to be elucidated.

Methods and Results

Wild-type or apolipoprotein E knockout male C57BL/6J background mice were fed standard chow for 16, 32, and 52 weeks, and the morphology of the aortic arch, descending aorta, and abdominal aorta was compared. Atheromatous plaque formation progressed with age, particularly in the aortic arch and abdominal aorta but not in the descending aorta. In addition, we found that the numbers of macrophages, T-lymphocytes, and microvessels, assessed by anti-F4/80, CD3, and CD31 antibodies, were higher in the adventitia of the abdominal aorta at 52 weeks. These numbers were positively correlated with plaque formation, but negatively correlated with elastin content, resulting in the enlargement of the total vessel area. In aortic tissues, interleukin-6 levels increased in the atheromatous plaque with age, whereas the level of regulated on activation, normal T cell expressed and secreted (RANTES) increased with age, and compared with other sites, it was particularly distributed in inflammatory cells in the adventitia of the abdominal aorta.

Conclusion

This study suggests that adventitial inflammation contributes to the age-dependent structural alterations, and that the activation/inactivation of cytokines/chemokines is involved in the process.     

A Novel Control System for Polymerase Chain Reaction Using a RIKEN GS384 Thermalcycler

We have developed a novel high-throughput thermalcycler, theRIKEN GS384, which has a maximum of 1536 wells and whose temperaturecan be controlled accurately and simultaneously for a very smallvolume of a reaction mixture. In practice, the reaction is carriedout using four 384-well (3.5 mm in diameter) plate formats whichcan be automatically moved using a robotic arm. To achieve accuratetemperature control with high thermo-conductivity, we adoptedTeflon-coated aluminum well plates closely sandwiched betweensilicon sheet-covered lids on top and a graphite sheet below.The lids were kept at a higher temperature (2 to 5°C) thanthe reaction wells. The temperature of the 1536 sample wellswas controlled accurately without temperature variability amongthe wells or evaporation, even for samples of very small volume(minimum 2 µl). We also developed a new type of plateformat which is similar to the 384-well place in terms of platesize, shape, and material, but which differs in the number (1536)and size (1.6 mm in diameter) of the wells. Since the amplificationreactions could be done precisely as well, a total of 6144 reactionscan potentially be carried out simultaneously using the GS384thermalcycler. This is very promising for DNA microfabricationtechnology. This thermalcycler offers the advantage of high-throughputDNA analysis which should be useful for DNA diagnoses or forthe human genome project.