First Report of Rhizoctonia solani on Mung Bean (Vigna radiata) Sprouts in California.

In March 2007, mung bean (Vigna radiata) sprouts produced in an indoor sprouting facility in northern California developed brown lesions beginning 5 days after germination. Dark brown-to-reddish brown lesions with distinct margins developed on the stem, hypocotyl, and first true leaves of affected sprouts. Although all seed is routinely soaked in 20,000 mg Ca(OCl)2/liter of water for 15 min before germination, approximately 5 to 10% of the bean sprouts in several growing baskets (1.5 × 1.5 m) were affected and had to be discarded. Each basket contained approximately 1 t of sprouts. To isolate the causal organism, symptomatic stems were surface disinfested for 1 min in 0.5% NaOCl and incubated on acidified potato dextrose agar (PDA) at 25°C. Cultures were identified as Rhizoctonia solani on the basis of morphological features including right-angled branching of brown hyphae and the presence of sclerotia. PCR amplification of the internal transcribed spacer region was performed with primers RS1 and RS4 (2). Sequences were identical to R. solani AG4-HG-II in GenBank (Accession No. AF354074). To conduct pathogenicity tests, a 5-mm2-diameter disk from the margin of a culture of the fungus on PDA was placed in the center of 25 5-day-old germinated sprouts placed in a plastic box (15 × 10 × 5 cm) held at 25°C. Two isolates of R. solani cultured from different lots of sprouts were included in the assays. Controls received noncolonized agar. Treatments were replicated four times and each experiment was repeated three times. A moist paper towel was included in each box to maintain humidity. After 3 days, symptoms developed in the inoculated boxes but not in the noninoculated boxes. The fungus was reisolated from lesions, completing Koch's postulates. To our knowledge, this is the first report of R. solani on mung bean sprouts in a commercial sprouting facility. However, R. solani has been associated with root rot of mung bean plants in the field (1). References: (1) T. R. Anderson. Can. Plant Dis. Surv. 65:1, 1985. (2) C. Guillemaut et al. Can. J. Microbiol. 49:556, 2003.

Molecular analysis of a major antigenic region of the 240-kD protein of Mi-2 autoantigen.

Anti-Mi-2 autoantibody is strongly associated with dermatomyositis and found in sera of 20% of patients. Mi-2 antigen contains at least eight components and previous evidence suggested that the 240-kD protein was the antigenic component for at least some sera. In this study, anti-M-2 patient sera were used to screen human thymocyte and HeLa cell lambda gt11 expression libraries, and two clones from each had plaques specifically reactive with anti-Mi-2 sera. Studies with affinity-purified antibody supported the identification of the clones. All of 44 anti-Mi-2 sera reacted with the plaques, but none of 44 control sera reacted significantly. The cDNAs were identical, and full sequencing of one revealed an open reading frame spanning a 1,054-bp insert. Rescreening the library with the cDNA yielded a 1,589-bp cDNA that continued the open reading frame. The Mi-2 cDNA hybridized to a single 7.5-8.0 kb mRNA of HeLa cells, by Northern blot. Rabbit antiserum directed at a portion of the cDNA product reacted with HeLa 240-kD Mi-2 protein. The sequence was notable for four potential zinc-fingers and several charged regions. The protein encoded by the cDNA produced in vitro reacted with only one of five of the Mi-2 sera. These findings indicate that the Mi-2 240 kD is a novel protein that is antigenic for all Mi-2 sera, and strongly suggests that a major common epitope is conformational in nature.

Regulation of the gp80 and gp130 subunits of the IL-6 receptor by sex steroids in the murine bone marrow.

Both estrogen and androgen exert their antiosteoporotic effects, at least in part, by inhibiting IL-6 production, thereby suppressing osteoclastogenesis. Several observations, however, suggest that besides increased IL-6 production, sensitivity of the osteoclastogenic process to this cytokine is altered after ovariectomy. Based on this and evidence that the ligand-binding subunit of the IL-6 receptor (gp80) is a limiting factor for the actions of IL-6 on bone, we hypothesized that sex steroids regulate expression of the IL-6 receptor as well. We report that 17beta-estradiol or dihydrotestosterone in vitro decreased the abundance of the gp80 mRNA as well as the mRNA of the signal-transducing subunit of the IL-6 receptor (gp130) in cells of the bone marrow stromal/osteoblastic lineage, and also decreased gp130 protein levels. These effects did not require new protein synthesis. In contrast to sex steroids, parathyroid hormone stimulated gp130 expression; this effect was opposed by sex steroids. Consistent with these findings, ovariectomy in mice caused an increase in expression of gp80, gp130, and IL-6 mRNAs in ex vivo bone marrow cell cultures as determined by quantitative reverse transcription (RT)-PCR, and confirmed on an individual cell basis using in situ RT-PCR. The demonstration of increased expression of the IL-6 receptor after loss of sex steroids provides an explanation for why IL-6 is important for skeletal homeostasis in the sex steroid-deficient, but not replete, state.

Classical genotropic versus kinase-initiated regulation of gene transcription by the estrogen receptor alpha.

Elucidation of kinase-initiated routes by which the estrogen receptors alpha and beta (ERalpha and ERbeta) control gene transcription, along with evidence of distinct biologic outcomes in response to ligands that can selectively activate nongenotropic signaling of the ERs or the androgen receptor, suggest that the ERs control a range of genes wider than that regulated by their direct association with DNA. To ascertain the extent and significance of nongenotropic ER-mediated transcription, we employed transduced HeLa cells expressing wild-type ERalpha or the ligand binding domain of ERalpha localized to the cell membrane (E-Mem), the OB-6 osteoblastic cell line, MCF-7 breast carcinoma cells and uteri from mice treated with 17beta-estradiol (E(2)), or the nongenotropic signaling activator 4-estren-3alpha,17beta-diol (estren). E(2) and estren induced ERK1/2 and Akt phosphorylation in ERalpha or E-Mem stably transfected HeLa cells; however, the phosphorylation kinetics differed between the two cell lines. In all four models, nongenotropic ER actions regulated a population of genes distinct from those regulated by genotropic ER actions. Specifically, the expression of Wnt2, Frizzled10, Egr-1, and c-Fos was strongly up-regulated in E-Mem-containing HeLa cells treated with E(2) or estren, or in ERalpha-containing HeLa cells treated with estren. Up-regulation of Frizzled10 by estren was reproduced in MCF-7 cells. Egr-1 was up-regulated by both estren and E(2); but complement 3, only by E(2) in the uteri. Estren had no effect on complement 3, cathepsin D, progesterone receptor, bcl-2, and cyclin D1 in MCF-7 cells, whereas E(2) up-regulated all these estrogen response element or activating protein-1-containing genes. These results support an extensive divergence in gene expression depending on the mode of ER activation.

Glucocorticoids act directly on osteoclasts to increase their life span and reduce bone density.

Glucocorticoid administration to mice results in a rapid loss of bone mineral density due to an imbalance in osteoblast and osteoclast numbers. Whereas excess glucocorticoids reduce both osteoblast and osteoclast precursors, cancellous osteoclast number surprisingly does not decrease as does osteoblast number, presumably due to the ability of glucocorticoids to promote osteoclast life span. Whether glucocorticoids act directly on osteoclasts in vivo to promote their life span and whether this contributes to the rapid loss of bone with glucocorticoid excess remains unknown. To determine the direct effects of glucocorticoids on osteoclasts in vivo, we expressed 11beta-hydroxysteroid dehydrogenase type 2, an enzyme that inactivates glucocorticoids, specifically in the osteoclasts of transgenic mice using the tartrate-resistant acid phosphatase promoter. Bone mass, geometry, and histomorphometry were similar in untreated wild-type and transgenic animals. Glucocorticoid administration for 7 d caused equivalent increases in cancellous osteoblast apoptosis, and equivalent decreases in osteoblasts, osteoid, and bone formation, in wild-type and transgenic mice. In contrast, glucocorticoids stimulated expression of the mRNA for calcitonin receptor, an osteoclast product, in wild-type but not transgenic mice. Consistent with the previous finding that glucocorticoids decrease osteoclast precursors and prolong osteoclast life span, glucocorticoids decreased cancellous osteoclast number in the transgenic mice but not wild-type mice. In accord with this decrease in osteoclast number, the loss of bone density observed in wild-type mice was strikingly prevented in transgenic mice. These results demonstrate for the first time that the early, rapid loss of bone caused by glucocorticoid excess results from direct actions on osteoclasts.

Chronic elevation of parathyroid hormone in mice reduces expression of sclerostin by osteocytes: a novel mechanism for hormonal control of osteoblastogenesis.

Both chronic excess of PTH, as in hyperparathyroidism, and intermittent elevation of PTH (by daily injections) increase the number of osteoblasts; albeit, the former is associated with bone catabolism and the later with bone anabolism. Intermittent PTH increases osteoblast number by attenuating osteoblast apoptosis, an effect that requires the transcription factor Runx2. However, chronic elevation of PTH does not affect osteoblast apoptosis because it stimulates the proteasomal degradation of Runx2. Here, we studied the effects of PTH on Sost, a Runx2 target gene expressed in osteocytes (former osteoblasts embedded in the bone matrix), which antagonizes the pro-osteoblastogenic actions of bone morphogenetic proteins and Wnts. We report that continuous infusion of PTH to mice for 4 d decreased Sost mRNA expression in vertebral bone by 80-90%. This effect was accompanied by a comparable reduction of sclerostin, the product of Sost, in osteocytes, as determined by quantitative immunoblot analysis of bone extracts and by immunostaining. In contrast, a single injection of PTH caused a transient 50% reduction in Sost mRNA at 2 h, but four daily injections had no effect on Sost mRNA or sclerostin. PTH strongly decreased Sost expression in osteocytes formed in primary cultures of neonatal murine calvaria cells as well as in osteocytic MLO-A5 cells, demonstrating a direct effect of PTH on this cell type. These results, together with evidence that sclerostin antagonizes bone morphogenetic proteins and Wnts, strongly suggest that suppression of Sost by PTH represents a novel mechanism for hormonal control of osteoblastogenesis mediated by osteocytes.

Anti-CD45RB monoclonal antibody-mediated transplantation tolerance.

Currently, lifelong immunosuppression is required for organ transplant recipients. The majority of transplant recipients will eventually develop chronic rejection with resultant graft loss, despite treatment with powerful immunosuppressive agents. These agents are also associated with numerous toxicities including reduced immunity against infection and malignancy. Therefore, the central goal in transplant science is to devise tolerance strategies in an attempt to establish a state of prolonged non-reactivity against the allograft, accompanied with preservation of an intact immune system. Although predictable tolerance induction has been elusive, we found that short course of the novel immunomodulatory agent, anti-CD45RB monoclonal antibody, leads to indefinite acceptance of renal allografts in mice, and has been shown to markedly prolong allograft survival in primates. We review the current state of development of this antibody, and the progress made in defining its mechanism of action.

Essential requirement of BMPs-2/4 for both osteoblast and osteoclast formation in murine bone marrow cultures from adult mice: antagonism by noggin.

Bone morphogenetic proteins (BMPs) have been heretofore implicated in the induction of osteoblast differentiation from uncommitted progenitors during embryonic skeletogenesis and fracture healing. We have tested the hypothesis that BMPs are also involved in the osteoblastogenesis that takes place in the bone marrow in postnatal life. To do this, we took advantage of the properties of noggin, a recently discovered protein that binds BMP-2 and -4 and blocks their action. Addition of human recombinant noggin to bone marrow cell cultures from normal adult mice inhibited both osteoblast and osteoclast formation; these effects were reversed by exogenous BMP-2. Consistent with these findings, BMP-2 and -4 and BMP-2/4 receptor transcripts and proteins were detected in these primary cultures, in a bone marrow-derived stromal/osteoblastic cell line, as well as in murine adult whole bone; noggin expression was also documented in all these preparations. Moreover, addition of antinoggin antibody caused an increase in osteoblast progenitor formation. These findings suggest that BMP-2 and -4 are expressed in the bone marrow in postnatal life and serve to maintain the continuous supply of osteoblasts and osteoclasts; and that, in fact, BMP-2/4-induced commitment to the osteoblastic lineage is a prerequisite for osteoclast development. Hence, BMPs, perhaps in balance with noggin and possibly other antagonists, may provide the tonic baseline control of the rate of bone remodeling on which other inputs (e.g., hormonal, biomechanical, etc.) operate.

Association of hY4 pseudogenes with Alu repeats and abundance of hY RNA-like sequences in the human genome.

Three loci having homology with the small human cytoplasmic RNA, hY4, were isolated from human genomic DNA libraries and sequenced. Each sequence contains dispersed mismatches as compared with hY4 RNA, is followed by an A-rich or A + T-rich sequence, and is bordered by direct repeats. Each of these loci, therefore, appears to constitute a small RNA class-III pseudogene. Surprisingly, two of the three loci are associated with Alu repeats. In the hY4.B7 locus, the hY4 sequence has integrated into the tail of an Alu element and in the hY4.F2 locus, an Alu sequence has inserted into the hY4 tail, confirming that A-rich tracts are preferential targets for retroposition. In addition, Southern blots with probes for each of the four hY RNAs indicate that hY RNA-like sequences are abundant in the human genome.

Y3 is the most conserved small RNA component of Ro ribonucleoprotein complexes in vertebrate species.

YRNAs are small cytoplasmic RNAs that are components of the Ro ribonucleoprotein complex. This complex, which also includes the 60-kDa Ro protein, is a human autoantigen which is conserved among vertebrates, and is of unknown function. Multiple sequences with YRNA homology, known as YRNA-like sequences, have been detected in rabbit, mouse, duck, iguana and frog genomes with human Y cDNA probes. As judged by Northern blots of total RNA, however, not all of these genomic YRNA-like sequences are expressed. Complementary DNA and putative gene sequences for iguana Y3 (iY3) and iguana Y4 (iY4) Ro RNAs have been determined and used, along with previously sequenced human and frog Ro YRNA sequences, to construct the most likely Y3 and Y4 RNA secondary structures. The data presented indicate that Y3 is the most conserved Ro RNA, not only by its more consistent presence in other species, but also at the levels of sequence divergence and secondary structure similarity. The differences observed between the secondary structure solutions for the Y3 and Y4 Ro RNAs are consistent with the possibility that these RNAs perform different cellular functions.

Cbfa1 does not regulate RANKL gene activity in stromal/osteoblastic cells.

The rates of osteoblast and osteoclast formation are tightly balanced, possibly due to the requirement of mesenchymal osteoblast progenitors for osteoclastogenesis. Osteoblast differentiation requires the transcription factor Cbfa1, whereas osteoclastogenesis results from the interaction between receptor activator of NF kappa B ligand (RANKL), expressed on stromal/osteoblastic cells, and RANK, a surface receptor on hematopoietic precursors. A striking decrease in the number of osteoclasts in Cbfa1-deficient mice suggested that Cbfa1 might be involved in RANKL expression. To investigate this possibility and to elucidate the mechanisms regulating RANKL expression, we isolated the 5'-flanking region of the murine RANKL gene and found that it contains two potential binding sites for Cbfa1 (OSE2-like sites). Cbfa1 bound to either of these sites in gel shift assays and stimulated the activity of a chimeric promoter consisting of multimerized RANKL OSE2-like sites inserted upstream from a minimal thymidine kinase (tk) promoter in transient transfections. However, Cbfa1 cotransfection did not stimulate murine RANKL promoter-luciferase constructs. Further analysis revealed that removal of these sites from the RANKL promoter by either site-directed mutagenesis or 5'-deletion did not alter the basal activity of promoter-reporter constructs. Conditional expression of Cbfa1 in a stromal/osteoblastic cell line stimulated osteocalcin mRNA by fivefold, but had no significant effect on RANKL mRNA levels. Conversely, conditional expression of a dominant-negative form of Cbfa1 in the same cell line inhibited osteocalcin mRNA by threefold, but had no effect on RANKL mRNA. Although these results cannot rule out a novel function for Cbfa1 in RANKL expression, they demonstrate that Cbfa1 does not regulate RANKL gene activity in the same manner as known targets of this transcription factor, such as osteocalcin.

A possible role for the 60-kD Ro autoantigen in a discard pathway for defective 5S rRNA precursors.

The Ro autoantigen is a 60-kD protein that is usually found in small cytoplasmic RNA-protein complexes known as Ro RNPs. Although the Ro RNPs are abundant and conserved components of a variety of vertebrate and invertebrate cells, their function is unknown. We have discovered that the Ro protein is also found complexed with certain variant 5S rRNAs in Xenopus oocytes. These RNAs contain one or more point mutations compared with the major oocyte 5S rRNA sequence as well as additional nucleotides at the 3' end. We demonstrate that the Ro protein binds specifically mutant 5S rRNAs containing 3' terminal extensions. These mutant RNAs are processed inefficiently to mature 5S rRNA and most eventually are degraded. The observation that the Ro autoantigen specifically associates with defective 5S rRNA precursors suggests that this protein may function as part of a novel quality control or discard pathway for 5S rRNA production.

Xenopus Ro ribonucleoproteins: members of an evolutionarily conserved class of cytoplasmic ribonucleoproteins.

Ro small ribonucleoproteins consist of a 60-kDa protein and possibly additional proteins complexed with several small RNA molecules. The RNA components of these particles, designated Y RNAs, are about 100 nt long. Although these small ribonucleoproteins are abundant components of a variety of vertebrate species and cell types, their subcellular location is controversial, and their function is completely unknown. We have identified and characterized the Ro RNPs of Xenopus laevis. Three of the four distinct Xenopus Y RNAs appear to be related to the previously sequenced human hY3, hY4, and hY5 RNAs. The fourth Xenopus Y RNA, xY alpha, does not appear to be a homologue of any of the human Y RNAs. Each of the human and Xenopus Y RNAs possesses a conserved stem that contains the binding site for the 60-kDa Ro protein. Xenopus and human 60-kDa Ro proteins are 78% identical in amino acid sequence, with the conservation extending throughout the entire protein. When human hY3 RNA is mixed with Xenopus egg extracts, the human RNA assembles with the Xenopus Ro protein to form chimeric Ro ribonucleoproteins. By analyzing RNA extracted from manually enucleated oocytes and germinal vesicles, we have determined that Y RNAs are located in the oocyte cytoplasm. By examining the distribution of mouse Ro ribonucleoproteins in cytoplast and karyoplast fractions derived from L-929 cells, we have determined that Ro ribonucleoprotein particles also primarily reside in the cytoplasm of mammalian cells.

Isolation and characterization of the human gp130 promoter. Regulation by STATS.

Glycoprotein 130 (gp130), a shared component of all the receptors for the interleukin-6 cytokine family, transduces cytokine signals in part by activating latent cytoplasmic signal transducers and activators of transcription (STATs). STATs subsequently translocate into the nucleus and stimulate gene expression. In the studies reported here, the 5'-flanking region of the human gp130 gene was isolated and the transcription initiation sites were mapped. To demonstrate that the isolated DNA fragment contained a functional promoter, a plasmid construct containing 2433 base pairs of the gp130 5'-flanking region, inserted upstream from the firefly luciferase gene, was transiently transfected into HepG2 hepatoma cells. The construct exhibited constitutive promoter activity. In addition, a 5-h treatment with interleukin-6 or oncostatin M stimulated the activity of this promoter severalfold. Localization of the cytokine response element by 5'-deletion analysis and site-directed mutagenesis revealed a cis-acting binding site for activated STAT complexes. Furthermore, DNA binding analysis demonstrated that this element binds activated STAT1 and STAT3 homo- and heterodimers. This STAT-binding element was sufficient to confer cytokine stimulation to a minimal herpesvirus thymidine kinase promoter. These results establish that the DNA fragment we have isolated contains the human gp130 promoter and that interleukin-6 type cytokines may influence the activity of this promoter via activated STATs.

A subset of hY RNAs is associated with erythrocyte Ro ribonucleoproteins.

The Ro autoantigen is a mammalian cellular ribonucleoprotein (RNP) of unknown function. We have demonstrated that hY1 and hY4 Ro RNAs are associated with erythrocyte Ro RNPs and represent a subset of the four hY RNAs found in HeLa cell and leukocyte Ro RNPs. We have cloned and sequenced hY4 RNA, the only hY RNA not sequenced previously, from a polymerase chain reaction amplified erythrocyte hY cDNA library. Sequencing of the erythrocyte hY RNAs in conjunction with Northern blot analysis confirms that the erythrocyte hY RNAs contain the same sequences as the respective HeLa cell RNAs of similar mobility. Ribonuclease inhibition activity has been found in erythrocytes and this activity inhibits the degradation of hY3 and hY5 in leukocyte lysates thereby favoring the possibility that the presence of hY1 and hY4 in erythrocytes is the result of differential expression of the hY RNAs in erythrocyte precursors.

Expression levels of gp130 in bone marrow stromal cells determine the magnitude of osteoclastogenic signals generated by IL-6-type cytokines.

Interleukin-6 (IL-6)-type cytokines stimulate osteoclast formation by activating the glycoprotein 130 (gp130) receptor subunit on stromal/osteoblastic cells, which in turn leads to signal transducer and activator of transcription 3 (STAT3)-mediated expression of receptor activator of NF-kappaB ligand (RANKL). Based on evidence that gp130 expression is regulated by a variety of cytokines and hormones, we have determined here whether changes in gp130 levels directly contribute to the magnitude of the osteoclastogenic stimulus delivered by IL-6-type cytokines. To accomplish this, gp130 protein levels were modulated using a tetracycline-regulated expression system in a stromal/osteoblastic cell line, UAMS-32, which supports osteoclast formation. Removal of doxycycline from the culture medium elevated gp130 expression and increased the responsiveness of a STAT-responsive promoter-luciferase construct to IL-6 complexed with its soluble receptor (IL-6+sIL-6R), but diminished the responsiveness to oncostatin M (OSM). IL-6+sIL-6R-stimulated osteoclast formation was greater when osteoclast precursors were cocultured with the cells expressing elevated gp130 levels than when cells expressing low gp130 levels were used. However, increased gp130 levels reduced OSM-stimulated osteoclast formation. These results establish that the level of gp130 in stromal/osteoblastic cells directly modulates the magnitude of the osteoclastogenic response to IL-6-type cytokines such that an increase in gp130 increases the cellular responsiveness to IL-6+sIL-6R but reduces responsiveness to OSM.

The Ro ribonucleoprotein particle: induction of autoantibodies and the detection of Ro RNAs among species.

High titers of autoantibody specific for the Ro(SSA) ribonucleoprotein are frequently found in patients with systemic lupus erythematosus and Sjogren's syndrome. In this study we have analyzed the immune responses to the Ro particle when utilized as an immunogen in animal hosts. Anti-Ro autoantibodies which bound autologous Ro ribonucleoprotein particles were induced in rabbits. In immunodiffusion studies using crude rabbit tissue extracts, the rabbit antibody made a precipitin line of identity with a prototype human anti-Ro serum. In solid-phase assays, the human autoimmune serum and the antigen-induced rabbit serum competed for similar or overlapping epitopes on the Ro particle. The rabbit and human sera precipitated the four Ro RNAs from human cells as well as four previously uncharacterized Ro RNAs from a bovine cell line, three Ro RNAs from a rabbit cell line, and two Ro RNAs from duck cells. While total numbers of cellular Ro RNAs differ among species, all possess an RNA of common size which comigrated with the hY1 of human cells.