The relationship between female rank and reproductive parameters of the ringtailed lemur: a preliminary analysis

We used data from a 13-year field study of wild ringtailed lemurs to analyze the relationship between female rank and reproductive parameters. In medium and small groups there were no significant differences in birth rate, infant mortality rate, and the number of surviving infants between the female rank categories. On the other hand, in large sized groups low-ranked females had a smaller number of surviving infants than middle-ranked females. This suggests that in large sized groups, within-group competition lowered the values of reproductive parameters of low-ranked females. On the other hand, high and low-ranked females of small sized groups tended to have a smaller number of surviving infants than high-ranked females of medium sized groups and middle-ranked females of large sized groups. Between-group competition should lower the values of their reproductive parameters. In sum, these results fit the expectation from Wrangham’s (1980) inter group feeding competition model.     

Molecular analysis of complement component C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus

 The amino acid sequences of the human terminal complement components show extensive structural similarity to each other. In this study the C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus, were cloned and analyzed. The derived deduced amino acid sequences of the two terminal components were homologous to those of humans, in that the sequences of both species contained LDL receptor, EGF precursor, and two thrombospondin domains. Japanese flounder C9 was found to have a second thrombospondin region in the C-terminus, similar to that reported for rainbow trout and pufferfish. Moreover, these two complement component cDNAs of Japanese flounder had partial similarity to human perforin. These findings show that Japanese flounder C8β and C9 have similar structures, which supports the hypothesis that the terminal complement genes originated from the same ancestral gene. Collectively, these features emphasize the strong similarity among the members of the terminal complement family. Received: 23 March 1999 / Revised: 1 June 1999     

Alteration of substrate specificity of aspartase by directed evolution

Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K(m) value and V(max) value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K(m) value and V(max) value for the l-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme.     

Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors

Paired Ig-like type 2 receptors (PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory (PILRalpha) and activating (PILRbeta) receptors. PILRs are widely expressed in immune cells and recognize the sialylated O-glycosylated ligand CD99, which is expressed on activated T cells, to regulate immune responses. To date, their biophysical properties have not yet been examined. Here we report the affinity, kinetic, and thermodynamic analyses of PILR-CD99 interactions using surface plasmon resonance (SPR) together with site-directed mutagenesis. The SPR analysis clearly demonstrated that inhibitory PILRalpha can bind to CD99 with low affinity (K(d) approximately 2.2 microm), but activating PILRbeta binds with approximately 40 times lower affinity (K(d) approximately 85 microm). In addition to our previous mutagenesis study (Wang, J., Shiratori, I., Saito, T., Lanier, L. L., and Arase, H. (2008) J. Immunol. 180, 1686-1693), the SPR analysis showed that PILRalpha can bind to each Ala mutant of the two CD99 O-glycosylated sites (Thr-45 and Thr-50) with similar binding affinity to wild-type CD99. This indicated that both residues act as independent and equivalent PILRalpha binding sites, consistent with the highly flexible structure of CD99. On the other hand, it is further confirmed that PILRbeta can bind the T50A mutant, but not the T45A mutant, indicating a recognition difference between PILRalpha and PILRbeta. Kinetic studies demonstrated that the PILR-CD99 interactions show fast dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses revealed that the PILRalpha-CD99 interaction is enthalpically driven with a large entropy loss (-TDeltaS = 8.9 kcal.mol(-1)), suggesting the reduction of flexibility upon complex formation. This is in contrast to the entropically driven binding of selectins to sugar-modified ligands involved in leukocyte rolling and infiltration, which may reflect their functional differences.     

Antimetastatic effect of endogenous tumor necrosis factor induced by the treatment of recombinant interferon γ followed by an analogue (GLA-60) to synthetic lipid A subunit

Summary We have investigated the effect of endogenous production of tumor necrosis factor (TNF) induced by the combination of recombinant interferon (rIFN) as a primer followed by GLA-60 as a trigger (rIFN/GLA-60) on murine lung metastases caused by B16-BL6 melanoma. In order to examine the therapeutic effect of endogenous TNF on tumor metastasis, the ability of multiple administrations of rIFN/GLA-60 to induce TNF production was also tested. The multiple administrations of rIFN/GLA-60 at intervals of 2 days were effective for the induction of endogenous TNF in mice but continuous multiple administrations of them for 2–4 days were not. In tumor-bearing mice, the production of endogenous TNF by rIFN/GLA-60 was less than that of normal mice, but treatment 3 days after the surgical excision of primary tumors showed the endogenous TNF production to be similar to that in normal mice. In the experimental lung metastasis model, intravenous administration of rIFN followed by intravenous or intranasal administration of GLA-60 showed potent inhibition of lung metastases of B16-BL6 melanoma, whereas the reverse sequence of administration (GLA-60/rIFN) or administration of a mixture of rIFN and GLA-60, which cannot induce the production of TNF, caused no inhibition of lung metastases. These results indicated that the regression of tumor metastases by rIFN/GLA-60 was mediated by the production of endogenous TNF in addition to the direct effects of both immunostimulants. Furthermore, the administration of rIFN and GLA-60 significantly inhibited the tumor metastases in spontaneous lung metastasis model. These results may provide a promising approach for the treatment of cancer metastasis as a result of its ability to induce endogenous TNF.     

Transforming growth factor beta2 is a neuronal death-inducing ligand for amyloid-beta precursor protein

APP, amyloid beta precursor protein, is linked to the onset of Alzheimer's disease (AD). We have here found that transforming growth factor beta2 (TGFbeta2), but not TGFbeta1, binds to APP. The binding affinity of TGFbeta2 to APP is lower than the binding affinity of TGFbeta2 to the TGFbeta receptor. On binding to APP, TGFbeta2 activates an APP-mediated death pathway via heterotrimeric G protein G(o), c-Jun N-terminal kinase, NADPH oxidase, and caspase 3 and/or related caspases. Overall degrees of TGFbeta2-induced death are larger in cells expressing a familial AD-related mutant APP than in those expressing wild-type APP. Consequently, superphysiological concentrations of TGFbeta2 induce neuronal death in primary cortical neurons, whose one allele of the APP gene is knocked in with the V642I mutation. Combined with the finding indicated by several earlier reports that both neural and glial expression of TGFbeta2 was upregulated in AD brains, it is speculated that TGFbeta2 may contribute to the development of AD-related neuronal cell death.     

Vitamin K2 and geranylgeraniol, its side chain component, inhibited osteoclast formation in a different manner

We comparatively examined the mechanism by which vitamin K(2) (Menatetrenone, MK4) and its side chain component, geranylgeraniol (GGO), inhibited osteoclast formation in the co-culture system of stromal cells with spleen cells. Both MK4 and GGO inhibited osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)). MK4, but not GGO, inhibited cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in the co-culture system. To elucidate the precise mechanism of the inhibitory effect of GGO on osteoclast formation, the co-cultured cells were stimulated with PGE(2). GGO, but not MK4, inhibited osteoclast formation via suppression of the receptor activator of NF-kappaB ligand (RANKL) expression. Moreover, GGO abolished the disruption of osteoclastic actin rings induced by nitrogen-containing bisphosphonate (N-BP), whereas MK4 did not affect it at all. These data suggest that MK4 inhibited osteoclast formation independently of GGO, and that MK4, but not GGO, has no competitive action on the anti-osteoporotic effect of N-BP.     

<Emphasis Type="Italic">Rol</Emphasis> (root loci) gene as a positive selection marker to produce marker-free <Emphasis Type="Italic">Petunia hybrida</Emphasis>

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring a rol-type MAT vector, pMAT101, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing rol gene as the selection marker gene. LacZ gene was used as a model gene of interest. Infected explants were cultured on plant growth regulator (PGR)- and antibiotic-free half-strength MS medium. Sixty-five percent of the infected explants produced hairy roots. The hairy roots were separated and proliferated on 1/2 MS hormone-free medium. Shoots produced from the hairy roots on 1/2 MS medium supplemented with benzylaminopurine (BA) and naphthalene acetic acid (NAA) exhibited hairy root syndrome (Ri syndrome) such as dwarfed, reduced apical dominance, short internodes and increased rooting, but subsequently produced normal-looking marker-free shoots. Molecular analysis of DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only LacZ gene, and the removable cassette consisting of rol, R (recombinase) and GUS genes was excised. From this study it can be concluded that the chimeric rol genes can be used as a selection marker for Agrobacterinum-mediated transformation of Petunia hybrida and that the production of marker-free normal transgenic plants is possible without using selective chemical agents employing rol-type MAT vector.