MOLECULAR DIVERGENCE AND CHARACTERIZATION OF TWO CHLOROPLAST DIVISION GENES,FTSZ1 AND FTSZ2, IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA)1

Two FtsZ paralogues (NbFtsZ1 and NbFtsZ2) were isolated from the unicellular green alga Nannochloris bacillaris Naumann. These sequences encoded proteins of 435 and 439 amino acids with tubulin signature motifs (GGGTG[T/S]G), which are important for GTP binding activity. NbFtsZ1 and NbFtsZ2 had four and three introns, respectively, and two different putative core promoters; a TATA box (TATAAAA) and an initiator element (CCCAGG) were located 40 bp and 80 bp upstream of the coding regions of NbFtsZ1 and NbFtsZ2, respectively. Southern blot hybridization and contour‐clamped homogeneous electric field electrophoresis showed that N. bacillaris contained at least one copy of each gene and that NbFtsZ1 was located on chromosome 5 and NbFtsZ2 on chromosome 3 or 4. Phylogenetically, NbFtsZ1 and NbFtsZ2 belong to the vascular plant protein families FtsZ1 and FtsZ2, respectively. The FtsZ1 proteins do not contain carboxy‐terminal consensus sequences, whereas all FtsZ2 proteins possess the consensus sequence (I/V)PxFL(R/K)(K/R)(K/R). Our study has shown that NbFtsZ2 possesses a similar consensus sequence (VPDFLRRK), whereas NbFtsZ1 does not, further supporting their classification as FtsZ2 and FtsZ1. Escherichia coli ftsZ mutants transformed with cloned NbFtsZ1, and NbFtsZ2 cDNAs were restored for the capacity to divide by binary fission, suggesting that the proteins retained the ability to function in the bacterium. An anti‐NbFtsZ2 antibody specifically recognized a single protein band of approximately 51 kDa on an immunoblot of N. bacillaris cellular proteins. Immunostaining of the algal cells with this antibody produced an intense fluorescent signal as a ring near the middle of the cell, which corresponded to the chloroplast division site.     

The reactivity of alpha-hydroxyhaem and verdohaem bound to haem oxygenase-1 to dioxygen and sodium dithionite.

Recently we have shown that ferric alpha-hydroxyhaem bound to haem oxygenase-1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem.274, 18196-18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al. have reported that the major reaction product of ferric alpha-hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous alpha-hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta1432, 203-213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. alpha-hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of alpha-hydroxyhaem with haem oxygenase may be small and a substantial amount of free alpha-hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air-sensitive and is therefore easily converted to a further oxidized species with excess O2. Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH-cytochrome P450 reductase.     

Morphological and histopathological changes in tongues of experimentally developed acromegaly-like rats.

An acromegaly-like rat model recently developed by exogenous administration of insulin-like growth factor I (IGF-I) was used to investigate morphological and histopathological tongue changes and clarify whether the changes were reversible. Human recombinant IGF-I (640 microg/day) was continuously subcutaneously infused into ten-week-old male rats for four weeks (IGF-I group; n = 6). Control sham-operated animals were injected saline alone (control group; n = 6). Rats were sacrificed immediately on ending administration at the age of fourteen weeks. Another 12 rats (6 from each group) were housed for an additional four weeks after administration ended. Total IGF-I (human + rat) increased significantly during administration, returning to control levels afterwards. Tongue weights significantly increased with histopathological changes present (increases in the muscle-bundle width, spaces between muscle-bundles and epithelium thickness) in the IGF-I group compared to control rats. Tongue size returned to control levels after discontinuation of IGF-I administration. These findings suggest that the characteristic tongue enlargement was developed experimentally in our acromegaly-like rat model, and that such morphological and histopathological tongue changes are reversible on normalization of circulating IGF-I levels.     

Wolbachia Infections in the Cimicidae: Museum Specimens as an Untapped Resource for Endosymbiont Surveys

Wolbachia spp. are obligate maternally inherited endosymbiotic bacteria that infect diverse arthropods and filarial nematodes. Previous microscopic and molecular studies have identified Wolbachia in several bed bug species (Cimicidae), but little is known about how widespread Wolbachia infections are among the Cimicidae. Because cimicids of non-medical importance are not commonly collected, we hypothesized that preserved museum specimens could be assayed for Wolbachia infections. For the screening of museum specimens, we designed a set of primers that specifically amplify small diagnostic fragments (130 to 240 bp) of the Wolbachia 16S rRNA gene. Using these and other previously published primers, we screened 39 cimicid species (spanning 16 genera and all 6 recognized subfamilies) and 2 species of the sister family Polyctenidae for Wolbachia infections using museum and wild-caught material. Amplified fragments were sequenced to confirm that our primers were amplifying Wolbachia DNA. We identified 10 infections, 8 of which were previously undescribed. Infections in the F supergroup were common in the subfamily Cimicinae, while infections in the A supergroup were identified in the subfamilies Afrocimicinae and Haematosiphoninae. Even though specimens were degraded, we detected infections in over 23% of cimicid species. Our results indicate that Wolbachia infections may be common among cimicids and that archived museum material is a useful untapped resource for invertebrate endosymbiont surveys. The new screening primers listed in this report will be useful for other researchers conducting Wolbachia surveys with specimens with less-than-optimum DNA quality.     

Identification of conservation measures to protect the Japanese endangered plant species Aster kantoensis

To identify the factors responsible for degrading the habitat of the endangered plant species Aster kantoensis, as well as the vulnerable life stage where this occurs, we carried out sowing experiments. Two natural habitats were simulated, being situated along the floodplains of the Tama River in central Japan. Seeds collected from a natural habitat were sown in two apparently suitable locations (Tomoda and Ishida sites). Germination, survival, growth, and seed production were subsequently monitored from 1993 through to 1997. The Tomoda site was a gravel bar in floodplains formed by flooding in 1991, while the Ishida site (two plots) was one gravel bar where several plants were growing sparsely and another where a population had become extinct in 1992. Seed cohorts completed their life cycle within 3 years at the Ishida site and within 5 years at the Tomoda site. Monitored parameters at Ishida were substantially lower than those at Tomoda. In addition, estimates of population growth indicated an increase at Tomoda and a rapid decrease at Ishida. However, degradation of habitats seemed to occur at Tomoda over the monitored periods. In view of our results, we conclude that natural germination of about 0.13% is needed for increasing population size. The major factors for decreasing population size are believed to be the lack of safe sites for germination and seedling establishment in old habitats (>10years). Conservation measures are suggested based on these findings.     

Production of Interleukin-1 by Primary Cultured Parenchymal Liver Cells (Hepatocytes)

The production of interleukin-1 (IL-1) by cultured parenchymal liver cells was revealed by a biological assay with an IL-1-dependent cell line, Northern blot analysis, and in situ hybridization. Inhibition experiments on the IL-1 activity with anti IL-1α antibody also support the presence of IL-1α in the supernatant of cultured parenchymal liver cells. Based on these results, we discuss the possibility of IL-1 production by parenchymal liver cells in vivo.     

Relationship between Hemolytic Activity and Adsorption Capacity of Aluminum Hydroxide and Calcium Phosphate as Immunological Adjuvants for Biologicals

Aluminum hydroxide (Al) and calcium phosphate (Ca) gels have been used as vaccine adjuvants for many years. We investigated mechanism of the hemolytic activities of both adjuvant materials. The hemolytic activity of each gel depended on the gel dose. The adsorption capacities and the hemolytic activities of both adjuvants decreased as the concentration of phosphate increased in a gel-washing solution. A positive correlation between the hemolytic activity and the adsorption capacity was found in Al-gel. A disruptive effect of Ca-gel on membrane of erythrocytes was shown by electron microscopy. Ca-gel required more than 10 times as much pre-adsorbed ovalbumin as did Al-gel to inhibit the hemolysis. These results suggest that the hemolytic activity of both adjuvant materials depended mainly on the adsorption ability, and it may be useful to control the adsorption ability of adjuvants to reduce their hemolytic activity.     

Effect of some constituents of chicken egg yolk lipoprotein on the growth and IgM production of human-human hybridoma cells and other human-derived cells

Chicken egg yolk lipoprotein (YLP) was partially fractionated into some constituents, and the effect of constituents of YLP were examined on the growth and immunoglobulin (IgM and IgG) secretion of a HB4C5 human-human hybridoma cell line cultured in serum-free medium. Among the fractions, YP-1 and YP-2 fractions (LDL-rich fractions) were found to enhance the growth and IgM secretion of HB4C5 cells. The promoting activity was found in the commercial LDL. The lipid fraction in YP-2 fraction conjugated with 2-maltosyl-a-cyclodextrin was found to enhance the growth and IgM secretion of HB4C5 cells. Livetin-rich YP-3 and YP-4 fractions had no significant promoting activity. Commercial -livetin and phosphatidyl choline possessed no growth-promoting activity. Phosphatidyl choline enhanced the IgM secretion of HB4C5 cells.