Silkworm arylsulfatase in the midgut content is expressed in the silk gland and fed via smearing on the food from the spinneret.

We found the activity of arylsulfatase in the midgut contents of the silkworm, Bombyx mori. We identified a 60-kDa protein that comigrates with the activity on a column chromatography following ammonium sulfate precipitation. Based on its partial amino acid sequence, we searched for its coding gene using Basic Local Alignment Search Tool (BLAST) and identified KWMTBOMO05106. Transcriptional data suggest a specific expression of the gene in middle silk glands. The majority (80%) of arylsulfatase activity was found in the silk glands, concurring the specific transcription in the silk gland. Observing the feeding behaviour of the silkworm, we found that silkworms smear a mucus secretes from the spinneret on the food pellet as they feed on. Arylsulfatase activity was also detected in the food pellet bitten by the silkworm as well as in the gut content. Furthermore, arylsulfatase activity was not detected either in the food pellet and in the gut content when silkworms had obstructed the spinneret. These results suggest that arylsulfatase is secreted from the silk glands and may contribute to digestive function.

Effects of molecular mass and hydrophobicity on transport rates through non-specific pathways of the silkworm larva midgut.

We previously reported that therapeutic drug effects in the silkworm infection model are largely influenced by midgut permeability. In this report, we describe the effects of drug molecular mass and hydrophobicity on transport through the silkworm larva midgut membrane. Hydrophilic compounds with a molecular mass of greater than 400Da did not permeate the silkworm larva midgut, and the hydrophobicity of similar-sized compounds had positive effects on the transport rate. Furthermore, we compared transport rates through the midgut membrane between cefcapene sodium (CFPN-Na) and cefcapene pivoxil (CFPN-PI), which is a CFPN-Na prodrug. The in vitro transport rate of CFPN-PI was three times faster than that of CFPN-Na. Moreover, when CFPN-PI and CFPN-Na were injected into the living silkworm larva midgut, CFPN-PI appeared rapidly in the haemolymph, whereas CFPN-Na did not. The 50% effective dose (ED50) of CFPN-PI administered via the midgut was one-sixth that of CFPN-Na. These findings suggest that the general features of the non-specific transport route are similar between silkworm larvae and mammals.

Duffy antigen is important for the lethal effect of the lethal strain of Plasmodium yoelii 17XL.

We studied the potential role of the Duffy antigen and glycophorin A as receptors for rodent malaria parasite invasion of erythrocytes. Parasitemia increased exponentially after infection with Plasmodium berghei NK65, P. chabaudi, and P. vinckei in Duffy antigen knockout, glycophorin A knockout, and wild-type mice, indicating that the Duffy antigen and glycophorin A are not essential for these malaria parasites. However, parasitemia of the Duffy antigen knockout mice infected with P. yoelii 17XL remained constant from day 5 to 14 after infection, and then decreased, resulting in autotherapy. The treatment of P. yoelii 17XL-infected Duffy antigen knockout mice with anti-CD4 antibody increased the parasitemia 15 days after infection and the mice eventually died, indicating that CD-4-positive cells play an important role in the clearance of P. yoelii 17XL at the late stage of the infection.

Quantitative evaluation of the therapeutic effects of antibiotics using silkworms infected with human pathogenic microorganisms.

The injection of bacteria (Staphylococcus aureus, Stenotrophomonas maltophilia) or true fungi (Candida albicans, Candida tropicalis) that are pathogenic to humans into the silkworm hemolymph leads to death of the larvae within 2 days. Antibiotics used for clinical purposes have therapeutic effects on silkworms infected with these pathogens. The 50% effective doses obtained by injection into the silkworm hemolymph are consistent with those reported for mice. Injection of vancomycin and kanamycin into the silkworm hemolymph was effective, but oral administration was not. Chloramphenicol, which is effective by oral administration, appeared in the silkworm hemolymph soon after injection into the midgut, whereas vancomycin did not. Isolated midgut membranes were impermeable to vancomycin. Thus, the ineffectiveness of oral administration of vancomycin to silkworms is due to a lack of intestinal absorption.

Glycophorin A requirement for expression of O-linked antigens on the erythrocyte membrane.

BACKGROUND: Glycophorin A (GPA) has a large number of sialic acid-containing oligosaccharide chains. GPA is highly conserved among vertebrates, mice with a GPA deletion have not been reported and GPA's physiologic role remains uncertain. RESULTS: GPA-/- homozygotes were obtained by intercrossing GPA+/- heterozygotes based on Mendelian genetics. The amount of O-linked oligosaccharide chains in the erythrocyte membrane of GPA-/- mice decreased to 60% compared to that of the wild-type mice. Flow cytometry and Western blot analysis revealed that the TER antigen that is associated with GPA on the erythrocyte membrane was totally abrogated from the cell surface in GPA-/- mice. Several glycoproteins that were detected with peanut agglutinin (PNA), a lectin that recognizes O-linked oligosaccharide chains, were absent from the GPA-/- erythrocyte membrane. Erythrocytes lacking GPA were more sensitive to hypo-osmotic stress than wild-type erythrocyte. CONCLUSIONS: GPA-/- mice show apparently normal phenotypes at least during the early generations. The disappearance of many glycoproteins recognized by PNA lectin on the GPA-/- erythrocyte membrane proteins suggests that GPA has an essential role in the expression of O-linked antigens on the erythrocyte membrane protein. These interactions of GPA and other glycoproteins may contribute to maintaining the physical strength of the erythrocyte membrane.

Induction of apoptosis by depletion of DNA topoisomerase IIalpha in mammalian cells.

Inactivation of topoisomerase (topo) IIalpha arrests murine embryonic development. In topo IIalpha-depleted embryos, nuclei were partitioned to daughter cells without complete separation and formed an interconnecting droplet-like structure. The present study examined the fates of topo IIalpha-depleted cells with the droplet-like nuclear structure. When the embryos with abnormal nuclei were further incubated, apoptosis was induced along with the formation of fragmented and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive nuclei. ICRF-193 treatment of embryos activated caspases. Apoptosis induced by ICRF-193 was suppressed by z-VAD-fmk, a caspase inhibitor, and pifithrin-alpha, a p53 inhibitor. Moreover, when mitosis was blocked by nocodazole, ICRF-193-induced nuclear abnormalities and apoptosis were abolished. These data suggest that cycling through the M-phase is essential for ICRF-193-induced apoptosis. Nuclear abnormalities similar to those of topo IIalpha-depleted embryos were induced in HeLa cells in which topo IIalpha was knocked down by transfection with short interfering RNA (siRNA) against topo IIalpha, followed by induction of apoptosis. Our results suggest that topo IIalpha-depleted cells with the droplet-like nuclear structure induce apoptosis, which is dependent on caspase and p53 activity during the G1 phase in mammalian cells.