Inhibited Nodule Formation Research Articles

Inhibition of osteoblast differentiation by tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) has a key role in skeletal disease in which it promotes reduced bone formation by mature osteoblasts and increased osteoclastic resorption. Here we show that TNF inhibits differentiation of osteoblasts from precursor cells. TNF-alpha treatment of fetal calvaria precursor cells, which spontaneously differentiate to the osteoblast phenotype over 21 days, inhibited differentiation as shown by reduced formation of multilayered, mineralizing nodules and decreased secretion of the skeletal-specific matrix protein osteocalcin. The effect of TNF was dose dependent with an IC50 of 0.6 ng/ml, indicating a high sensitivity of these precursor cells. Addition of TNF-alpha from days 2-21, 2-14, 7-14, and 7-10 inhibited nodule formation but addition of TNF after day 14 had no effect. Partial inhibition of differentiation was observed with addition of TNF on only days 7-8, suggesting that TNF could act during a critical period of phenotype selection. Growth of cells on collagen-coated plates did not prevent TNF inhibition of differentiation, suggesting that inhibition of collagen deposition into matrix by proliferating cells could not, alone, explain the effect of TNF. Northern analysis revealed that TNF inhibited the expression of insulin-like growth factor I (IGF-I). TNF had no effect on expression of the osteogenic bone morphogenic proteins (BMPs-2, -4, and -6), or skeletal LIM protein (LMP-1), as determined by semiquantitative RT-PCR. Addition of IGF-I or BMP-6 to fetal calvaria precursor cell cultures enhanced differentiation but could not overcome TNF inhibition, suggesting that TNF acted downstream of these proteins in the differentiation pathway. The clonal osteoblastic cell line, MC3T3-E1-14, which acquires the osteoblast phenotype spontaneously in postconfluent culture, was also studied. TNF inhibited differentiation of MC3T3-E1-14 cells as shown by failure of mineralized matrix formation in the presence of calcium and phosphate. TNF was not cytotoxic to either cell type as shown by continued attachment and metabolism in culture, trypan blue exclusion, and Alamar Blue cytotoxicity assay. These results demonstrate that TNF-alpha is a potent inhibitor of osteoblast differentiation and suggest that TNF acts distal to IGF-I, BMPs, and LMP-1 in the progression toward the osteoblast phenotype.

Expression of Vascular Endothelial Growth Factors and Their Receptors during Osteoblast Differentiation

Endochondral bone formation is regulated by systemically and locally acting growth factors. A role for vascular endothelial growth factor (VEGF) in this process has recently been proposed, because inactivation of VEGF inhibits endochondral bone formation via inhibition of angiogenesis. Despite the known effect of VEGF as specific endothelial growth factor, its effects on osteoblast differentiation have not been studied. We, therefore, examined the expression of VEGF-A, -B, -C, and -D and their receptors in a model of osteoblast differentiation using the mouse preosteoblast-like cell line KS483. Early in differentiation, KS483 cells express low levels VEGF-A, -B, and -D messenger RNA, whereas during mineralization, KS483 cells express high levels. In addition, expression of the VEGF receptors, VEGFR1, VEGFR2, and VEGF165R/neuropilin, coincided with expression of their ligands, being maximally expressed during mineralization. VEGF-A production during osteoblast differentiation was stimulated by insulin-like growth factor I that enhances osteoblast differentiation and was inhibited by PTH-related peptide that inhibits osteoblast differentiation. Furthermore, continuous treatment of KS483 cells with recombinant human VEGF-A stimulated nodule formation. Although treatment of KS483 cells with soluble FLT1, an agent that blocks binding of VEGF-A and -B to VEGFR1, did not inhibit nodule formation, this observation does not exclude involvement of VEGFR2 in the regulation of osteoblast differentiation. As it is known that VEGF-A, -C, and -D can act through activation of VEGFR2, other isoforms might compensate for VEGF-A loss. The expression pattern of VEGFs and their receptors shown here suggests that VEGFs play an important role in the regulation of bone remodeling by attracting endothelial cells and osteoclasts and by stimulating osteoblast differentiation.

Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation.

Endochondral bone formation is regulated by systemically and locally acting growth factors. A role for vascular endothelial growth factor (VEGF) in this process has recently been proposed, because inactivation of VEGF inhibits endochondral bone formation via inhibition of angiogenesis. Despite the known effect of VEGF as specific endothelial growth factor, its effects on osteoblast differentiation have not been studied. We, therefore, examined the expression of VEGF-A, -B, -C, and -D and their receptors in a model of osteoblast differentiation using the mouse preosteoblast-like cell line KS483. Early in differentiation, KS483 cells express low levels VEGF-A, -B, and -D messenger RNA, whereas during mineralization, KS483 cells express high levels. In addition, expression of the VEGF receptors, VEGFR1, VEGFR2, and VEGF165R/neuropilin, coincided with expression of their ligands, being maximally expressed during mineralization. VEGF-A production during osteoblast differentiation was stimulated by insulin-like growth factor I that enhances osteoblast differentiation and was inhibited by PTH-related peptide that inhibits osteoblast differentiation. Furthermore, continuous treatment of KS483 cells with recombinant human VEGF-A stimulated nodule formation. Although treatment of KS483 cells with soluble FLT1, an agent that blocks binding of VEGF-A and -B to VEGFR1, did not inhibit nodule formation, this observation does not exclude involvement of VEGFR2 in the regulation of osteoblast differentiation. As it is known that VEGF-A, -C, and -D can act through activation of VEGFR2, other isoforms might compensate for VEGF-A loss. The expression pattern of VEGFs and their receptors shown here suggests that VEGFs play an important role in the regulation of bone remodeling by attracting endothelial cells and osteoclasts and by stimulating osteoblast differentiation.

Inhibition of chondrocyte differentiation in vitro by constitutive and inducible overexpression of the c-fos proto-oncogene.

We have investigated the role of c-Fos in chondrocyte differentiation in vitro using both constitutive and inducible overexpression approaches in ATDC5 chondrogenic cells, which undergo a well-defined sequence of differentiation from chondroprogenitors to fully differentiated hypertrophic chondrocytes. Initially, we constitutively overexpressed exogenous c-fos in ATDC5 cells. Several stable clones expressing high levels of exogenous c-fos were isolated and those also expressing the cartilage marker type II collagen showed a marked decrease in cartilage nodule formation. To investigate further whether c-Fos directly regulates cartilage differentiation independently of potential clonal variation, we generated additional clones in which exogenous c-fos expression was tightly controlled by a tetracycline-regulatable promoter. Two clones, DT7.1 and DT12.4 were capable of nodule formation in the absence of c-fos. However, upon induction of exogenous c-fos, differentiation was markedly reduced in DT7.1 cells and was virtually abolished in clone DT12.4. Pulse experiments indicated that induction of c-fos only at early stages of proliferation/differentiation inhibited nodule formation, and limiting dilution studies suggested that overexpression of c-fos decreased the frequency of chondroprogenitor cells within the clonal population. Interestingly, rates of proliferation and apoptosis were unaffected by c-fos overexpression under standard conditions, suggesting that these processes do not contribute to the observed inhibition of differentiation. Finally, gene expression analyses demonstrated that the expression of the cartilage markers type II collagen and PTH/PTHrP receptor were down-regulated in the presence of exogenous c-Fos and correlated well with the differentiation status. Moreover, induction of c-fos resulted in the concomitant increase in the expression of fra-1 and c-jun, further highlighting the importance of AP-1 transcription factors in chondrocyte differentiation. These data demonstrate that c-fos overexpression directly inhibits chondrocyte differentiation in vitro, and therefore these cell lines provide very useful tools for identifying novel c-Fos-responsive genes that regulate the differentiation and activity of chondrocytes.

Autoregulation of soybean–Bradyrhizobium nodule symbiosis is controlled by shoot or/and root factors

Two strains of Bradyrhizobium japonicum were evaluated with five commercial cultivars of soybean (Clark, Crauford, Davis, Centaur, and Nessen) and one hypernodulating mutant NOD1-3. The hypernodulating NOD1-3 produced 30–50 times the number of nodules of commercial cultivars either inoculated with B. japonicum strain USDA 123 or RCR 3409. Grafting of NOD1-3 shoots to Clark and Davis roots induced hypernodulation on roots of Clark and Davis but did not enhance nodulation when grafted onto the roots of Crauford, Centaur, and Nessen. In contrast, the shoots of Clark, Davis, Centaur and Nessen significantly inhibited nodule formation on the root of NOD1-3. However, Crauford shoots did not alter nodule formation on the roots of NOD1-3 as compared with self-grafts of NOD1-3. It appears that the shoot of NOD1-3 has the ability to alter autoregulatory control of nodulation of Clark and Davis cultivars, but not of Crauford, Centaur and Nessen. The results suggest that the regulation of nodulation in soybean cultivars Clark and Davis is controlled by the shoot factors, while the Crauford was root controlled. Reciprocal grafts between NOD1-3 and Centaur or Nessen indicate that both shoot and root factors are involved in regulation of nodulation. The results suggested that the regulation of nodulation did not depend on bradyrhizobial strains. The shoot control of hypernodulation may be causally related to differential root isoflavonoid levels, which are also controlled by shoot. Application of daidzein significantly enhanced the nodulation and nitrogenase activity of soybean cv. Clark. Root control of restricted nodulation of soybean cv. Centaur did not respond to the addition of daidzein in nutrient solution indicating that this character is not related to isoflavonoids. Therefore, autoregulation in Clark and Centaur plants may be separate events in legume–rhizobia symbiosis and regulated by different kinds of signals.

Osteoprogenitor cells in cell populations derived from mouse and rat calvaria differ in their response to corticosterone, cortisol, and cortisone

Osteoprogenitors present in cell populations derived from fetal or newborn rat and mouse calvaria differentiate in long term culture and form osteoblastic bone-forming colonies (bone nodules). Previous reports have indicated considerable differences between bone cell populations derived from these two species with regard to their proliferation in response to glucocorticoids. In the present investigation, we have focused on proliferation and differentiation of osteoprogenitor cells in these bone cell populations and evaluated the effect of corticosterone, the principal glucocorticoid of both mouse and rat. Cells were isolated by sequential collagenase digestion from calvaria of newborn (2–5 days) CD-1 mice [mouse calvariae (MC) cells] and term fetal Wistar rats [rat calvaria (RC) cells] and cultured for up to 25 days in α-minimal essential medium containing 10% fetal bovine serum (FBS), antibiotics, 50 μg/mL ascorbic acid, and 8–10 mmol/L β-glycerophosphate. In agreement with previous observations by us and others, corticosterone increased cell growth in RC cell cultures, but inhibited cell growth in MC cultures. In RC cell cultures, corticosterone (1–1000 nmol/L) increased the nodule number in a dose-dependent manner ( p < 0.001 for all concentrations above 3 nmol/L) with a maximal effect at 300 and 1000 nmol/L (threefold increase over control). In MC cells, on the other hand, corticosterone (0.3–1000 nmol/L) increased the nodule number only at 30 nmol/L (50%, p < 0.01) but inhibited nodule formation by 33% ( p < 0.001) at 1000 nmol/L. In both RC and MC cultures a linear relationship was found between the number of cells plated and number of nodules formed. When cultures were treated with cortisol (30–300 nmol/L), similar effects were observed; the number of nodules dose dependently increased in RC cell cultures and dose dependently decreased in MC cell cultures. Significantly, however, the inactive glucocorticoid cortisone also increased bone nodule formation in RC cell cultures and decreased bone nodule formation in MC cell cultures. Carbenoxolone, which blocks 11 β hydroxysteroid dehydrogenase and thus prevents conversion of cortisone to cortisol, partially inhibited the cortisone-induced effects on bone nodule formation in both RC and MC cell cultures, indicating that both RC and MC cells can convert inactive glucocorticoids to active metabolites. In conclusion, our results show that the glucocorticoids corticosterone and cortisol inhibit proliferation and differentiation of osteoprogenitors in MC cell cultures but stimulate these processes in rat-derived osteoprogenitors.

The Effects of Standard and Low Molecular Weight Heparin on Bone Nodule Formation In Vitro

Previously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.

Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro.

We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.

Temporal expression of PDGF receptors and PDGF regulatory effects on osteoblastic cells in mineralizing cultures.

Platelet-derived growth factor (PDGF) is mitogenic and chemotactic for osteoblastic cells in vitro. It is expressed during osseous wound healing and stimulates formation of new bone in vivo. PDGF stimulates cells by binding to specific cell surface receptors. The purpose of this study was to examine the effects of PDGF on osteoblastic proliferation and differentiation in long-term mineralizing cultures. Utilizing Northern blot analysis, we found that continuous PDGF treatment increased histone expression, indicative of enhanced proliferation, but suppressed osteoblast differentiation, demonstrated by inhibition of alkaline phosphatase, type I collagen, and osteocalcin expression. The inhibitory effect of PDGF on the differentiated function of osteoblasts was further established by findings that PDGF significantly inhibited nodule formation. The expression of PDGF receptors varied at different stages of culture. PDGF receptor mRNA expression increased when the cells had achieved a mature phenotype, during the stage of matrix maturation, and then decreased. However, as demonstrated by thymidine incorporation assays, the capacity of PDGF to stimulate DNA synthesis actually decreased during osteoblast maturation, as receptor expression increased. To investigate this apparent contradiction, tyrosyl phosphorylation and immunoblot assays were performed to assess changes in PDGF activation of their cognate receptors. The pattern of PDGF-induced tyrosyl phosphorylation remained relatively constant. This suggests that the diminished mitogenic activity of PDGF that occurs after osteoblast differentiation is regulated at a postreceptor level.

Fibronectin regulates calvarial osteoblast differentiation

The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.

Single-Leaf Plantlet Bioassays for the Study of Root Morphogenesis and Rhizobium-Legume Nodulation

New plant bioassays have been developed to (a) study root morphogenesis in small seeded legumes, and (b) enable the routine examination of large numbers of independently transformed plants. In addition, these bioassays enable analysis of specific plant gene expression. The single leaf plantlet assay uses simplified plants that are without shoot meristems, have a photosynthetic organ, grow roots which can be nodulated by Rhizobium bacteria, and can form lateral roots. The rooted cotyledon assay (RCA) was developed to investigate root morphogenesis of clover plants. Studies showed that early in the development of the root there was a cotyledon-derived inhibitor that could inhibit the continued growth of Rhizobium-induced nodule primordia. However, the activity of this inhibitor diminished with continued growth and maturation of the root. Rhizobia that can synthesise wild-type Nod factors can partially overcome this inhibition of nodule formation, the Nod signals acting as 'anti-cotyledon inhibitors'. Comparison of the RCA plantlets with normal seedlings suggests that the rooted cotyledons have a slightly slower root maturation, which enables the experimentalist time to probe mechanisms underlying root development. The rooted leaf assay was developed so that the symbiotic and physiological properties of large numbers of transgenic plants could be studied. This is a rapid technique that can use either untransformed or recently transformed plant material, and avoids the potential problem of a disturbed hormone balance that is inherent in the use of 'hairy roots' generated by the use of the Agrobacterium rhizogenes-mediated transformation of small-seeded legumes.

Pleiotropic effect of fluoranthene on anthocyanin synthesis and nodulation of Medicago sativa is reversed by the plant flavone luteolin.

The symbiosis between leguminous plants and soil bacteria of the genus Rhizobium is of considerable agronominal importance. Recently it has been found, that polycyclic aromatic hydrocarbons (PAHs; e.g. anthracene, phenanthrene, fluoranthene), occurring as ubiquitous environmental contaminants can inhibit nodulation of Medicago sativa. Fluoranthene is one of the dominant PAHs found in urban particulate matter, sewage sludge or beside motorways. Several organisms have been shown to be able to metabolize and mineralize fluoranthene but the uptake of fluoranthene is limited due to low solubility of fluoranthene in water and strong adsorption to humic substances in soil. Rhizobium meliloti cannot degrade fluoranthene. Toxic effects of fluoranthene on bacterial growth have never been observed. In contrast to their rhizobial symbiotic partners, alfalfa plants grown on a solidified fluoranthene-containing medium, exhibited symptoms of toxicity. They showed a dose-responsive decrease in shoot length and, if inoculated with R. meliloti, inhibition of nodule formation. Growth retardation is accompanied by a decrease in anthocyanin pigmentation of shoots, and an atypical accumulation of anthocyanins in roots. Plant flavonoids are known to play a central role in the signal exchange of the Legume-Rhizobium symbiosis. Phenylpropane derived compounds and flavonoids have been implicated in nodule development. Since fluoranthene impairs nodulationmore » and induces the production of anthocyanins, it is possible that these events are causally linked via phenylpropanoid metabolism. These experiments attempt to overcome the inhibitory effects of fluoranthene by exogeneous application of the flavonoid luteolin. This paper demonstrates that luteolin antagonizes the fluoranthene mediated inhibition of nodule formation and prevents the accumulation of anthocyanins in roots. 29 refs., 4 figs., 1 tab.« less

Interleukin-11 inhibits bone formation in vitro.

The effects of interleukin-11(IL-11) on the differentiation of osteoblast precursors was tested using a bone nodule forming assay in rat calvaria cell cultures. IL-11 caused a dose dependent inhibition of nodule formation, with 500 U/ml IL-11 resulting in complete inhibition of nodule formation. IL-11 also caused a reduction in alkaline phosphatase expression in these cultures. These effects are similar to, but more potent than, the actions of IL-6 on these cells. These results indicate that IL-11 is an osteotropic cytokine and suggest that IL-11 may be an important inhibitor of bone formation in health and disease.

Aluminum inhibits bone nodule formation and calcification in vitro.

Cells isolated from neonatal mouse calvariae can be induced to form mineralized nodules after exposure to ascorbic acid and beta-glycerophosphate. To determine whether aluminum inhibits nodule formation and subsequent mineralization, cells isolated from neonatal mouse calvariae were induced to form nodules and incubated with increasing concentrations of aluminum (10(-7) to 10(-5) M). Compared with control and 10(-7) M aluminum-supplemented cultures, the number of nodules formed and the number of nodules calcified were reduced in cells incubated with 10(-6) and 10(-5) M aluminum. The cumulative net calcium uptake into the nodules and their final calcium content were also decreased with 10(-6) and 10(-5) M aluminum. After 10 and 18 days of incubation, aluminum did not affect DNA synthesis or release of alkaline phosphatase but significantly inhibited collagen production. Thus aluminum induced a dose-dependent inhibition of nodule formation and calcification that may be related to its inhibition of collagen production.

Characterization of the 1,25-(OH)2D3-induced inhibition of bone nodule formation in long-term cultures of fetal rat calvaria cells.

We investigated the effects of 1,25-dihydroxyvitamin D3[1,25-(OH)2D3], on osteoprogenitor cell differentiation and bone nodule formation at various stages of differentiation by evaluating the effects on long term cultures of fetal rat calvaria (RC) cells. RC cells were plated at 3 x 10(4) cells/35-mm dish in alpha-minimal essential medium containing 15% fetal bovine serum, ascorbic acid, and beta-glycerophosphate (beta-GP), conditions under which bone nodules form. 1,25-(OH)2D3 inhibited bone nodule formation in a dose-dependent manner with total inhibition occurring at 1-10 nM and half-maximal inhibition occurring at approximately 0.06 nM. 1,25-(OH)2D3 also significantly stimulated RC cell growth in a dose-dependent manner in both the presence and absence of ascorbic acid. Addition of 1 nM 1,25-(OH)2D3 at different times after the start of culture inhibited nodule formation when added before and up to the early multilayering stage (up to day 11 of culture), but had no effect on nodule number when added later. When 1,25-(OH)2D3 was added at the start of the culture period and removed at the early multilayering stage, nodule formation was also inhibited. Pulses of 48-h duration also inhibited nodule formation, with maximal effect occurring between days 3 and 11. Thus, 1,25-(OH)2D3 inhibited osteoprogenitor cell differentiation during the earlier stages of culture before visible bone nodule formation occurred and the effect was not reversible upon removal of 1,25-(OH)2D3. In cultures grown to the multilayering stage in medium without ascorbic acid and beta-GP and then changed to medium with ascorbic acid and beta-GP, 1,25-(OH)2D3 inhibited when present before, but not after, the addition of ascorbic acid and beta-GP. Two other vitamin D3 metabolites, 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] and 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3] had inhibitory effects similar to 1,25-(OH)2D3. The effects were dose dependent for each metabolite tested and correlated with the biological effectiveness of these metabolites in other systems: i.e. 1,25-(OH)2D3 was more effective than 1,24,25-(OH)3D3 which in turn was more effective than 24,25-(OH)2D3. The data show that 1,25-(OH)2D3 inhibits osteoprogenitor cell differentiation at an early stage and at a time during which cell growth is stimulated.

Assimilation of15N2and15NO3−by Partially Nitrate-Tolerant Nodulation Mutants of Soybean

Growth-chamber studies were conducted to evaluate nitrogen assimilation by three hgpernodulated soybean [Glycine max (L.) Merr.] mutants (NOD1-3, NOD2-4, NOD3-7) and the Williams parent. Seeds were inoculated at planting and transplanted at day 7 to nutrient solution with 1 mol m -3 urea (optimizes nodule formation) or 5 mol m -3 NO 3 - (inhibits nodule formation). At 25 d after planting, separate plants were exposed to 15 N 2 or 15 NO 3 - for 3 to 48 h to evaluate N 2 fixation and NO 3 - assimilation

Nitrate Inhibition on Nodule Growth and Activity and Reversal of Inhibition by Indole Acetic Acid in Mungbean (Vigna radiata L.) Inoculated with Hup+ and Hup-Rhizobium Strains1

Continuous supply of nitrate in the nutrient solution for 40 d inhibited nodule formation and nitrogenase (acetylene reduction) activity more in mungbean (Vigna radiata L.) plants inoculated with hydrogen uptake negative (Hup-) Rhizobium strain M11 compared to plants inoculated with hydrogen uptake positive (Hup+) strain S24. Nitrate even at a low concentration of 1 mM inhibited nodule forsmation and nitrogenase activity (per plant) by 57 and 46% in plants inoculated with Hup- strain, and by 34 and 20% in plants inoculated with Hup+ strain, respectively. At 10 mM nitrate concentration, however, nodule formation was almost completely depressed. Addition of IAA (10-6 M) before inoculation alleviated but did not completely reversed nitrate (4 mM) inhibition on nodulation process and nitrogenase activity in plants inoculated with Hup- strain. However, in plants inoculated with Hup+ strain the same concentration of IAA not only reversed inhibitory effect of nitrate (4 mM) but further stimulated nodulation and per plant nitrogenase activity by 20 and 65%, respectively. In vivo activity of leaf NR increased as the concentration of nitrate increased in the nutrient solution. These observations suggest that diversion of photosynthates towards nitrate assimilation may be responsible for depressing nodule growth and function and, in this situation Hup system might. be supporting nitrogen fixation. Further, a role for IAA in nodule biogenesis has been indicated.

Nitrate Inhibition of Root‐Nodule Symbiosis in Doubly Rooted Soybean Plants

Split‐root experiments have demonstrated that NO−3 inhibits nodule formation on legumes primarily as a root‐localized effect rather than as a function of whole‐plant N nutrition. The degree to which the root‐localized effect is coupled with the N status of the shoot is not well understood. To investigate this, doubly rooted soybean [Glycine max (L.) Merr.] plants were produced by layering stems in adjacent pots to induce the formation of adventitious roots; thus, the effects of N added to the entire original root could be examined in terms of nodulation on both the original and the adventitious roots. When NO−3 level was sufficiently high to completely suppress nodulation on the original root, there was profuse nodulation and significant nitrogenase (C2H2 reduction) activity on the adventitious root. This indicated that even when mineral N supply to the root was abundant there was an N deficit in the shoot. The apparent poor coupling of N status of the root with that of the shoot was confirmed when doubly rooted plants accumulated more N than did singly rooted plants. Stem nodules of sesbania (Sesbania rostrata Brem. &amp; Oberm.) are analogous to adventitious root nodules of doubly rooted soybean in that they form on root primordia not in contact with added N; the adverse effects of applied NO−3 on nodule development and function were more pronounced on sesbania stems than on soybean adventitious roots. Therefore, the coupling of root‐N status with shoot‐N status may be particularly loose in soybean with potentially important implications for growth in N‐rich soils.

Alleviation of nitrate inhibition of soybean nodulation by high inoculum does not involve bacterial nitrate metabolism

Inoculation of soybean (Glycine max. cv. Bragg) plants with high level inoculum partially alleviated the nitrate inhibition of nodule formation (3 to 4 fold), but not nodule growth. This alleviation did not require the bacterial nitrate reductase asBradyrhizobium japonicum mutant strains 110CR1 and 110CR2 (both lacking assimilatory nitrate reductase activity) gave the same results as the wild type parent 311b110. The study was carried out in the glasshouse, thereby confirming preliminary field data by Herridgeet al. (1984) using a wild type bacterial inoculant.

Effects of insecticides on acetylene reduction by alfalfa, red clover and sweetclover

Seven organophosphate and carbanate insecticides were tested for their effects on C 2H 2-reduction by alfalfa ( Medicago sativa L.), sweetclovcr ( Melilotus alba L.) and red clover ( Trifolium pratense L.). Plants were grown in vitro for 21 days on nonstcrile vermiculite or soil substrates. Oftanol ® was inhibitory to most plants on both soil and vermiculite. Carbofuran's inhibitory effect was almost entirely limited to soil-grown plants. Carbaryl, aldicarb, chlorpyrifos, and terbufos were inhibitory to certain plants under some conditions. Phosmet was the least toxic. Significant differences were observed in toxicity which were dependent on the growth substrate used. The effects of carbamates tended to be restricted to plants grown on soil substrates rather than on vermiculite. Loss of C 2H 2-reducing ability was usually correlated with inhibition of nodule formation. The insecticides did not inhibit growth of Rhizobium meliloti or R. trifolii in broth culture.