Cytokine-specific induction of the TGF-beta inducible early gene (TIEG): regulation by specific members of the TGF-beta family

Select members of the TGF-beta family of cytokines play key regulatory roles in skeletal development, structure, and turnover. This laboratory has previously reported that TGF-beta treatment of immortalized normal human fetal osteoblast (hFOB) cells results in the rapid induction of the mRNA levels of a TGF-beta inducible early gene (TIEG) followed by changes in cell proliferation and bone matrix protein production. Previous studies have also shown that nonmembers of the TGF-beta superfamily showed little or no induction of TIEG mRNA. This article further addresses the cytokine specificity of this TIEG induction by examining whether activin and select bone morphogenetic proteins, (BMP-2, BMP-4, and BMP-6), which are representative of different subfamilies of this superfamily, also induce the expression of TIEG in hFOB cells. However, TGF-beta remained the most potent of these cytokines, inducing TIEG mRNA steady-state levels at 0.1 ng/ml, with a maximum induction of 24-fold at 2.0 ng/ml. The BMP-2 (16-fold), BMP-4 (4-fold), and activin (1-3-fold) also induced TIEG mRNA levels, but at reduced degrees compared to TGF-beta (24-fold), and only at much higher cytokine concentrations, e.g., 50-100 ng/ml, compared to 2 ng/ml for TGF-beta. BMP-6 showed no effect on TIEG mRNA levels. The TIEG protein levels generally correlated with the mRNA steady-state levels. As with TGF-beta, BMP-2 treatment of hFOB cells was shown by confocal microscopy to induce a rapid translocation of the TIEG protein to the nucleus. In summary, the relative potencies of these TGF-beta family members to induce TIEG expression generally follows the general osteoinductive capacity of these cytokines, with TGF-beta > BMP-2 > BMP-4 > activin > BMP-6.     

Variation in Efficacy of Isolates of the Fungus ARF Against the Soybean Cyst Nematode Heterodera glycines

An unnamed fungus, designated ARF, that parasitizes eggs and sedentary stages of cyst nematodes is a potential biological control agent of Heterodera glycines. The objectives of this study were to determine whether ARF isolates differ in their ability to suppress nematode numbers in soil and to compare the efficacy of ARF in heat-treated and native soil. The effectiveness of 11 ARF isolates was compared by introducing homogenized mycelium into heat-treated soil. Soybean seedlings were transplanted into pots containing fungus-infested soil and inoculated with H. glycines. After 30 or 60 days, the number of nematodes and the percentage of parasitized eggs were determined. Three isolates (907, 908, and TN14), which were previously reported to be weak egg parasites in vitro, consistently suppressed nematode numbers by 50% to 100%. Of the isolates previously reported to be aggressive egg parasites, four (903, BG2, MS3, and TN12) reduced nematode numbers by 56% to 69% in at least one experimental trial, but the other four had no effect on nematode numbers. When the efficacy of isolate TN14 was tested in heat-treated and native soil, nematode suppression was greater in the heat-treated soil in only one of two trials. In both soil treatments, nematode numbers were reduced by more than 60%. We conclude that virulence toward nematode eggs in vitro is a poor indicator of effectiveness of an ARF isolate in soil, and that the presence of soil microbes may reduce, but does not completely inhibit, activity of isolate TN14.     

Effect of temperature on seed production in the invasive grass Glyceria maxima (Hartm.) Holmb. (Poaceae) in South Africa

Temperature is one of the main factors that determine sexual reproduction in terrestrial and emergent aquatic plant species. The effect of temperature on sexual reproduction and seed production of Glyceria maxima (Hartm.) Holmb. in the southern hemisphere is unknown. Glyceria maxima collections in February 2010 at three isolated infestations in KwaZulu-Natal failed to yield a single seed, only empty panicles. Laboratory experiments showed that vernalisation had no consistent effect on seed production. Field- and laboratory-grown plants produced seeds in the 2010/2011 season, because of having sufficient time at optimum temperatures required for seed production (1 491 and 1 585 hours, respectively), compared to a shorter period (1 352 hours) of suitable temperatures during the 2009/2010 growing season. An inadequate period of optimum temperatures (15–25°C) during seed production resulted in the lack of seeds in the field in the 2009/2010 growing season. This study showed that temperature and duration of exposure thereto during the seed-production period play vital roles in G. maxima sexual reproduction.     

Regulation of osteoclastogenesis and RANK expression by TGF-beta1

Transforming growth factor-beta (TGF-beta) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-beta on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-kappaB ligand (RANK-L) (50 ng/ml), TGF-beta1 (0.01-5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-beta1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-beta1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-beta1 resulted in a significant increase in the percentage of RANK+ RAW cells (P < 0.05), as well as an increase in the fluorescence intensity per cell (P < 0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-beta directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D- galactofuranosidase inhibitors

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D- galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4- aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.