Oral immunization with Escherichia coli K-12 of the fifth instar larvae of the silkworm, Bombyx mori, reared on an artificial diet under completely aseptic conditions.

1. Effect of oral administration of live or formalin-treated Escherichia coli (E. coli) K-12 to the fifth instar, days 1 and 3 larvae of the silkworm, Bombyx mori, on induction of antibacterial activity in the haemolymph was investigated using the silkworms reared on an artificial diet under completely aseptic conditions. 2. When live E. coli was administered to the male day 1 larvae, low but significant antibacterial activity of 3.8 mm was detectable in the haemolymph of one individual at 48 hr after immunization. The proportion of the larvae to express antibacterial activity increased thereafter and at 120 hr after immunization, all three individuals showed antibacterial activity. In day 3 male larvae, activity was detectable at 48 and 72 hr after immunization. 3. When formalin-treated E. coli was orally administered to days 1 and 3 male larvae, no activity was detectable at any time post-immunization. 4. In the second experiment, when day 1 larvae, females and males were orally immunized with live E. coli, only females showed antibacterial activity in the haemolymph, beginning from 24 hr after immunization and up to 96 hr. 5. Removal of an antibiotic, chloramphenicol, from ingredients of an artificial diet was required for induction of antibacterial activity with oral administration of live E. coli. 6. When live E. coli that grows at pH 9.0 was selected and used for oral immunization, antibacterial activity was induced both in females and males at 72 hr after immunization and the activity was observed at 96 hr. 7. These results suggest that establishment of oral immunization with live E. coli in the silkworm larvae requires multiplication of E. coli in the midgut lumen and possibly its colonization on the luminal surface.     

Retrotransposon-mediated restoration of Chlorella telomeres: accumulation of Zepp retrotransposons at termini of newly formed minichromosomes

To elucidate the contribution of LINE-like retrotransposon Zepp elements to the formation and maintenance of chromosomal telomeres, newly formed minichromosomes in irradiated Chlorella vulgaris cells were isolated and structurally characterized. A minichromosome (miniV4) of ~700 kb in size contained a Zepp cluster taking the place of the telomeric repeats on one terminus, whereas the other end of this chromosome consisted of canonical telomeric repeats. The Zepp copies in this cluster were in a tandem array with their poly(A) tails towards the centromere. Another minichromosome Y32 (~400 kb in size) was shown to have several copies of Zepp elements on both termini. On the right arm terminus, two copies of Zepp were found in a tandem array with poly(A) tracts facing towards the chromosomal end. The poly(A) tail and the 3′-end of ~400 bp of the distal copy were replaced by the telomeric repeats. On the 5′-side of the proximal copy was another Zepp element in the reverse orientation. These newly formed telomeric structures are very similar to those previously found in the left arm of chromosome I and the terminus of an unidentified chromosome and support the model of Zepp-mediated restoration and maintenance of Chlorella telomeres.     

Timing of the G1/S transition in tobacco pollen vegetative cells as a primary step towards androgenesis in vitro

Key message

Mid-bicellular pollen vegetative cells in tobacco escape from G1 arrest and proceed to the G1/S transition towards androgenesis within 1 day under glutamine starvation conditions in vitro.

Abstract

In the Nicotiana tabacum pollen culture system, immature pollen grains at the mid-bicellular stage can mature in the presence of glutamine; however, if glutamine is absent, they deviate from their native cell fate in a few days. The glutamine-starved pollen grains cannot undergo maturation, even when supplied with glutamine later. Instead, they undergo cell division towards androgenesis slowly within 10 days in a medium containing appropriate nutrients. During the culture period, they ought to escape from G1 arrest to proceed into S phase as the primary step towards androgenesis. However, this event has not been experimentally confirmed. Here, we demonstrated that the pollen vegetative cells proceeded to the G1/S transition within approximately 15–36 h after the start of culture. These results were obtained by analyzing transgenic pollen possessing a fusion gene encoding nuclear-localizing GFP under the control of an E2F motif-containing promoter isolated from a gene encoding one of DNA replication licensing factors. Observations using a 5-ethynyl-2′-deoxyuridine DNA labeling and detection technique uncovered that the G1/S transition was soon followed by S phase. These hallmarks of vegetative cells undergoing dedifferentiation give us new insights into upstream events causing the G1/S transition and also provide a novel strategy to increase the frequency of the androgenic response in tobacco and other species, including recalcitrants.     

Induction of antibacterial activity in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 in the anterior and posterior body part of the ligated larvae.

1. Induction of antibacterial activity was investigated in the ligated fifth instar larvae of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 into the haemocoel in the anterior and in the posterior body part, followed by activity determination by inhibition zone assay of the haemolymph at 12 and 24 hr after immunization. 2. At 12 hr after immunization, high antibacterial activity, approximately 6.8-7.5 mm in the anterior body part and 4.5-6.4 mm in the posterior body part in diameter of a clear zone (2.0 mm for no activity) was detectable in day 3 larvae. This result was in good contrast to expression of lectin gene in the ligated flesh fly, Sarcophaga peregrena (Shiraishi and Natori, 1988, FEBS Lett. 232, 163-166), in which only the anterior part of insect responded to stimulus of injury. 3. Antibacterial activity at 24 hr after immunization in days 3 or 4 ligated larvae was lower than that at 12 hr; 4.0-4.5 mm of activity was observed in the anterior body part, and no activity was observed in day 3 ligated larvae in the posterior body part. 4. Acid polyacrylamide gel electrophoresis of the haemolymph of immunized insects followed by overlay assay showed that the size of antibacterial activity bands were similar between the haemolymph from 12 hr and from 24 hr, and between the anterior and the posterior body part. This result was contradictory to the observation of activity by inhibition zone assay. The activity bands were associated with peptides that were similar to cecropin-like peptides A and B in the silkworm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced DNase I sensitivity of the rearranged c-myc gene in somatic cell hybrids between murine plasmacytoma cells and fibroblasts

In mouse plasmacytoma (MPC) S194, the rearranged c-myc gene was much more sensitive to DNase I digestion than the nonrearranged gene. The sensitivity of the rearranged c-myc was markedly reduced to the same extent as that of the nonrearranged one in hybrids between the MPC cells and the fibroblasts, but not in a hybrid between the MPC and the spleen cells. These results suggest that trans-acting factors in fibroblasts alter the DNase I-sensitive structure of the rearranged c-myc gene.     

Neutrophil attractant/activation protein-1 and monocyte chemoattractant protein-1 in rabbit. cDNA cloning and their expression in spleen cells

Rabbit neutrophil attractant/activation protein-1 (NAP-1) and monocyte chemoattractant protein-1 (MCP-1) were investigated. Rabbit spleen cells stimulated with 5 micrograms/ml of Con A produced both neutrophil and monocyte chemotactic activity. Physicochemical characteristics of those activities obtained by HPLC gel filtration and HPLC chromatofocusing were very similar to those of human NAP-1 and MCP-1, suggesting that rabbit spleen cells produce NAP-1 and MCP-1 after Con A stimulation. A cDNA library was constructed from mRNA purified from Con A-stimulated rabbit spleen cells and screened with oligonucleotide probes. By two rounds of screening, NAP-1 and MCP-1 cDNA were cloned. NAP-1 cDNA comprises 1500 bp with an open reading frame that encodes for a 101-amino acid protein highly similar to human NAP-1. MCP-1 cDNA comprises 607 bp with an open reading frame that encodes for a 124-amino acid protein highly similar to human MCP-1. Expression of NAP-1 and MCP-1 mRNA by rabbit spleen cells was studied. Both Con A- and LPS-stimulated spleen cells expressed NAP-1 and MCP-1 mRNA, but the kinetics of expression were different. Con A rapidly induced high NAP-1 and MCP-1 mRNA expression. LPS also rapidly induced NAP-1 mRNA expression, but high MCP-1 mRNA expression was not observed until 15 h after stimulation. Immunoprecipitation of metabolically labeled NAP-1 and MCP-1 with anti-human NAP-1 or MCP-1 polyclonal antibodies was attempted. Immunoprecipitated rabbit NAP-1 with a molecular mass of about 7 kDa was detected by SDS-PAGE and radioautography, but MCP-1 was not. Cloned rabbit NAP-1 and MCP-1 will give us opportunities to study the role of NAP-1 and MCP-1 in vivo.     

Significant increase in plasma 4β-hydroxycholesterol concentration in patients after kidney transplantation

Several previous studies have shown that renal failure decreases not only renal elimination but also metabolic clearance of drugs, particularly those metabolized by CYP3A. However, whether recovery of renal function results in recovery of hepatic CYP3A activity remains unknown. In this study, we evaluated the effect of renal function on CYP3A activity after kidney transplantation in patients with end-stage renal disease (ESRD) by measuring the change in CYP3A activity using plasma concentration of 4β-hydroxycholesterol as a biomarker. The study enrolled 13 patients with ESRD who underwent the first kidney allograft transplantation. Morning blood samples were collected before and 3, 7, 10, 14, 21, 30, 60, 90, 120, 150 and 180 days after kidney transplantation. Plasma concentration of 4β-hydroxycholesterol was measured using GC-MS. Compared with before kidney transplantation, creatinine clearance increased significantly from day 3 after kidney transplantation and stabilized thereafter. Plasma concentration of 4β-hydroxycholesterol was elevated significantly on days 90 and 180 after kidney transplantation. In conclusion, this study suggests the recovery of CYP3A activity with improvement in renal function after kidney transplantation in patients with ESRD.     

Heat coma and its relationship to ocean dynamics in the oceanic sea skaters of Halobates (Heteroptera: Gerridae) inhabiting Indian and Pacific Oceans

This study was performed to clarify how weather and current dynamics affect the resistance to temperature change in the oceanic sea skaters, Halobates. Heat coma temperature (HCT) was measured for the adults and 5th instar larvae of four Halobates species collected from a fixed sampling location (12°00′N, 135°00′E ) in western tropical Pacific Ocean and from 13 locations in the eastern area of the India Ocean ranging from 08°00′N-06°00′S and 86°00-76°00′E. Both the gap temperature for heat coma (GTHC, mean±SD: 7.83±1.86 °C, n=32) and the heat coma temperature (HCP, 35.03±1.80 °C, n=32) of individuals collected from the Pacific Ocean, during the first half (10 days) of the sampling period at the fixed sampling point, were significantly higher than those during the second half (GTHC: 5.10±2.05 °C, n=63; HCP: 34.03±2.02 °C, n=63). The reduction in heat tolerance shown in the second half of the 20 day period may have been caused by a decrease in air temperature due to rainfall that occurred around the sampling point accompanied with the arrival of Typhoon No. 6.In the study of individuals collected from the Indian Ocean, significantly higher average GTHCs of >8 °C were recorded for the adult H. micans collected at 02°00′S and 06°00′S (89°00′E) than those at 0°00-8°00′N in the eastern Indian Ocean. Dynamic mixture of water from northern and southern currents occurs at 02°00-6°00′S of the Indian Ocean and might relate to such high heat tolerance.Temperature dynamics in the ocean habitat might directly affect the temperature resistance of the oceanic sea skaters.     

Surface plasmon resonance biosensor detects the downstream events of active PKCbeta in antigen-stimulated mast cells

Surface plasmon resonance (SPR) biosensors detect large changes of angle of resonance (AR) when RBL-2H3 mast cells are cultured on a sensor chip and stimulated with antigen. However, the detail of molecular events that are responsible for such large changes of AR remained unknown. In this study, we investigated the relationship between intracellular signaling events induced by antigen and the change of AR, by genetic manipulation of intracellular signaling molecules; spleen tyrosine kinase (Syk), src-like adaptor protein (SLAP), linker for activation of T cells (LAT), growth-factor-receptor-bound protein 2 (Grb2), Grb2-related adaptor protein (Gads), and isotypes of protein kinase C (PKC). RBL-2H3 mast cells overexpressing dominant-negative Syk or SLAP, which both interfere with active Syk, exhibited only minimal increase of AR in response to antigen stimulation. Likewise, the interference of the activation of LAT and Gads, by expressing dominant-negative LAT and Gads, respectively, resulted in nearly complete suppression of the antigen-induced increase of AR. The cells overexpressing PKCs, apart from PKCbeta, showed a reduced extent of increase of AR in response to antigen stimulation. Moreover, the introduction of the small interfering RNA targeted against PKCbeta suppressed the antigen-induced increase of AR. These results indicate that the activation of Syk, LAT, Gads, and subsequent PKCbeta is indispensable for the antigen-induced increase of AR of mast cells detected by SPR biosensors.