Spectroscopic and structural investigations on modafinil by FT-IR, FT-Raman, NMR, UV-Vis and DFT methods.

Chiral sulfoxide based smart drug modafinil were studied experimentally and theoretically. Vibrational spectra were recorded in the mid IR region and electronic spectra were recorded in UV-Visible region. The molecular geometry, vibrational spectra, magnetic spectra and electronic spectra were simulated using Density Functional Theory (DFT) employed with B3LYP/6-311++G(d,p) basis set. The molecular geometry optimization, vibrational frequencies, chemical shifts and solvent effect on electronic properties were reported. The intermolecular interactions have been studied by Hirshfeld surface analysis. There is good agreement was found between calculated and observed values, thereby to confirm the molecular structure of modafinil.

Identification and pharmacological characterization of a novel inhibitor of autotaxin in rodent models of joint pain.

OBJECTIVE: Autotaxin is a secreted lysophospholipase that mediates the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA), a bioactive lipid mediator. Autotaxin levels in plasma and synovial fluid correlate with disease severity in patients with knee osteoarthritis (OA). The goal of this study was to develop and characterize a novel small molecule inhibitor of autotaxin to inhibit LPA production in vivo and determine its efficacy in animal models of musculoskeletal pain. DESIGN: Compound libraries were screened using an LPC coupled enzyme assay that measures the amount of choline released from LPC by the action of autotaxin. Hits from this assay were tested in a plasma assay to assess inhibition of endogenous plasma autotaxin and subsequently tested for their ability to lower plasma LPA levels upon oral dosing of rats. The best compounds were then tested in animal models of musculoskeletal pain. RESULTS: Compound screening led to the identification of compounds with nanomolar potency for inhibition of autotaxin activity. Studies in rats demonstrated a good correlation between compound exposure levels and a decrease in LPA levels in plasma. The leading molecule (compound-1) resulted in a dose dependent decrease in joint pain in the mono-sodium iodoacetate (MIA) and meniscal tear models and a decrease in bone fracture pain in the osteotomy model in rats. CONCLUSION: We have identified and characterized a novel small molecule inhibitor of autotaxin and demonstrated its efficacy in animal models of musculoskeletal pain. The inhibitor has the potential to serve as an analgesic for human OA and bone fracture.

Frequency of undiagnosed psoriatic arthritis among psoriasis patients in Australian dermatology practice.

BACKGROUND: Psoriatic arthritis commonly develops in psoriasis patients and, if undiagnosed, can lead to potentially avoidable joint damage and an increased risk of comorbidity and mortality. Increased awareness of PsA symptoms among dermatologists provides an opportunity for earlier diagnosis, more timely therapy and prevention of disability. OBJECTIVE: To provide Australian epidemiological data on the frequency of undiagnosed PsA among psoriasis patients in dermatology practice, and to investigate the impact of psoriasis on quality of life and work productivity. METHODS: Nine tertiary centre dermatology practices enrolled patients presenting with plaque psoriasis and no prior rheumatologist-confirmed PsA diagnosis. Patients were screened using the Psoriatic Arthritis Screening and Evaluation (PASE) questionnaire and were referred to a rheumatologist for assessment of PsA status using CASPAR criteria if they had a PASE score ≥44. RESULTS: Based on the composite and sequential application of PASE and CASPAR criteria, undiagnosed PsA among psoriasis patients in this study is 9% [95% CI: 6, 12]. The PPV of PASE in this setting is 26% [95% CI: 19, 34]. Nail involvement and chronic large plaque psoriasis were identified as independent positive predictors of PsA, whereas scalp psoriasis was an independent negative predictor of PsA. Patients with moderate-to-severe psoriasis (PASI ≥15) had lower quality of life scores than patients with less severe psoriasis. CONCLUSION: In this study, the frequency of undiagnosed PsA in Australian dermatology practice was 9% among plaque psoriasis patients with no prior PsA diagnosis. Compared with psoriasis alone, the impact of undiagnosed PsA on health-related quality of life of psoriasis patients is substantial.

Iron nanotube-silica composite (ZVI-S-PCAT modified silica composite) preparation, characterization and application as a recyclable catalytic system for 5-membered ring organic transformations.

Iron nanotube silica composite have been synthesized and studied through particle-size analysis, FTIR, SEM-EDX, TEM, XRD, UV, VSM, TGA-DTA and XPS techniques. The application of nanoframeworks as sustainable recyclable catalytic systems has been observed for azole cyclic ring organic transformations. The good reaction yields and characterization through (1)H NMR, (13)C NMR and mass analysis support the performance of the nanoframeworks. We also present here the synthesis of two novel compounds. Also the prepared nanoframework has been observed to show soft magnetism which provides a scope to be used in sensing devices.

Selective Autooxidation of Ethanol over Titania-Supported Molybdenum Oxide Catalysts: Structure and Reactivity.

We study the selective catalytic oxidation of ethanol with air as a sustainable alternative route to acetaldehyde. The reaction is catalysed by molybdenum oxide supported on titania, in a flow reactor under ambient pressure. High selectivity to acetaldehyde (70%-89%, depending on the Mo loading) is obtained at 150 °C. Subsequently, we investigate the structure/performance relationship for various molybdenum oxide species using a combination of techniques including diffuse reflectance UV-visible, infrared, X-ray photoelectron spectroscopies, X-ray diffraction and temperature programmed reduction. As their surface density increases, the monomeric molybdenum oxide species undergo two-dimensional and three-dimensional oligomerisation. This results in polymolybdates and molybdenum oxide crystallites. Importantly, the ethanol oxidation rate depends not only on the overall molybdenum loading and dispersion, but also on the type of molybdenum oxide species prevalent at each surface density and on the domain size. As the molybdenum oxide oligomerisation increases, electron delocalisation becomes easier. This lowers the absorption edge energy and increases the reaction rate.

A short-term pharmacodynamic model for monitoring aggrecanase activity: injection of monosodium iodoacetate (MIA) in rats and assessment of aggrecan neoepitope release in synovial fluid using novel ELISAs.

OBJECTIVE: To develop a short-term in vivo model in rats, with an enzyme-linked immunosorbent assay (ELISA) readout for specific aggrecanase-cleaved aggrecan fragments, to facilitate testing of aggrecanase inhibitors. METHODS: Monosodium iodoacetate (MIA), a metabolic inhibitor, was injected into the right knee joint of male Lewis rats and the release of aggrecanase-cleaved fragments of aggrecan containing the NITEGE or ARGN neoepitope was measured in the synovial fluid at 7 days post MIA injection using novel ELISAs. The ELISAs utilize a commercial antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan, in combination with either an alpha-NITEGE antibody (NITEGE ELISA) or an alpha-ARGS/BC3 antibody (ARGS ELISA), to detect aggrecanase-cleavage of aggrecan within the interglobular domain (IGD). Aggrecan fragments present in in vitro digests, in cytokine-treated cartilage explant culture supernatants and in rat synovial fluid lavage samples were detected and quantified using the two ELISAs. Small molecule inhibitors of aggrecanase activity were dosed orally on days 3-7 to determine their ability to inhibit MIA-induced generation of the NITEGE and ARGN neoepitopes measured in the rat synovial fluid. RESULTS: The NITEGE assay was shown to specifically detect the N-terminal fragment of aggrecan comprising the G1 domain and the NITEGE neoepitope sequence. This assay can readily measure aggrecanase-cleaved bovine, human and rat aggrecan without the need for deglycosylation. The ARGS assay specifically detects C-terminal fragments of aggrecan comprising the ARGS/ARGN neoepitope and the G2 domain. Keratan sulfate (KS) residues of aggrecan interfere with this ELISA, and hence this assay works well with native rat articular cartilage aggrecan (that lacks KS residues) and with deglycosylated bovine and human aggrecan. Injection of MIA into the rat knee joints resulted in a time-dependent increase in the release of aggrecanase-cleaved aggrecan fragments into the synovial fluid and treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of the generation of these neoepitopes. CONCLUSIONS: We have established a short-term in vivo model in rats that involves measurement of synovial fluid biomarkers that are dependent on aggrecanase activity in the joint. The short duration of the model combined with the mechanistic biomarker readout makes it very useful for the initial in vivo screening of aggrecanase inhibitors prior to testing them in time and resource-intensive disease models of osteoarthritis (OA).

Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.

OBJECTIVE: Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. METHODS: The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. RESULTS: The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. CONCLUSIONS: We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

Combining etanercept with traditional agents in the treatment of psoriasis: a review of the clinical evidence.

Psoriasis is a chronic, systemic inflammatory disorder manifesting primarily in skin and potentially in joints, frequently necessitating treatment with conventional systemic therapies, phototherapy or biological agents. Patients with moderate to severe disease suffer a diminished quality of life, experience significant comorbidities and have a higher mortality. Although traditional treatments are effective in the short-term, their use is often limited by concerns over long-term toxicity, including end-organ damage and risk of malignancy. Combination therapy is a commonly used approach and is often more effective than any single agent. Lower doses of two treatments in combination can also minimize potential side effects from a single agent at higher doses. Etanercept is a recombinant human tumour necrosis factor (TNF)α receptor (p75) protein fused with the Fc portion of IgG1 that binds to TNFα. This article reviews the evidence on the efficacy and safety of etanercept in combination with methotrexate, acitretin, narrowband UVB and cyclosporin. The largest body of evidence assesses the combination with methotrexate, although evidence is available for the other combinations. Data suggest that although highly effective as monotherapy, etanercept in combination with a conventional systemic agent can enhance efficacy and allow drug sparing. Potentially, the combination may also result in faster treatment responses and permit safe transitioning from one systemic agent to another. Evidence to date suggests that these benefits can be achieved without significant additional toxicity, although long-term data on the efficacy and safety of the combination in psoriatic populations is limited and further evaluation is warranted.

Identification of signal transduction pathways and promoter sequences that mediate parathyroid hormone 1-38 inhibition of osteoprotegerin gene expression.

Osteoprotegerin (OPG), a secreted member of the tumor necrosis receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Parathyroid hormone (PTH), a potent inducer of osteoclast formation, suppresses OPG mRNA expression in vitro and in vivo. To determine the molecular basis of this inhibition, we analyzed the effects of PTH on the human OPG promoter (-5917 to +19) fused with beta-galactosidase reporter gene in stable and transient transfections into rat osteoblast-like UMR106 cells. The effect of PTH on OPG promoter expression was biphasic and concentration-dependent. PTH (1-100 nM) induced the transcriptional activity of the OPG promoter (1.7-fold) at 8 h followed by a gradual decrease with maximal inhibition (6.6-fold) at 24-48 h. To ascertain the signal transduction pathways mediating PTH (1-38) effects on OPG gene expression, we compared the effects of PTH with PTH analogs, parathyroid hormone-related protein 1-34 (PTHrP 1-34), forskolin, 3-isobutyl-1-methylxanthine (IBMX), dibutyryl cAMP, phorbol-12-myristate-13-acetate (PMA), thapsigargin and calcium ionophore A23187. PTH 1-31 and PTHrP 1-34, which stimulate the cAMP/PKA pathway, and other activators of cAMP/PKA, forskolin, IBMX, N(6), O(2')-dibityryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP), all elicited a similar biphasic response on OPG promoter expression. PTH analogs PTH 3-34 and PTH 7-34, that do not stimulate cAMP production, had no effect on OPG expression. In contrast, phorbol-12-myristate-13-acetate (PMA), an activator of PKC, stimulated OPG promoter expression, while thapsigargin and calcium ionophore A23187, which increase intracellular Ca(2+), showed a dose-dependent inhibition of OPG promoter expression. To delineate the promoter sequences that mediate the inhibitory effects of PTH on OPG transcription, we analyzed systematic deletions of the OPG promoter for responsiveness in transient transfection assays. The major inhibitory effects of PTH were localized to 391 bp (-372 to +19) of the proximal promoter. Deletions of the promoter region led to a complete loss of responsiveness. Taken together, these results demonstrate that the inhibitory effects of PTH on OPG are mediated at the transcriptional level through cis elements in the proximal promoter. The similar biphasic response of OPG to PTH, PTH 1-31, PTHrP 1-34, forskolin, IBMX and dibutyryl cAMP suggests that PTH regulates OPG transcription via activation of the cAMP/PKA signal transduction pathway.

Stimulation of osteoprotegerin (OPG) gene expression by transforming growth factor-beta (TGF-beta). Mapping of the OPG promoter region that mediates TGF-beta effects.

Transforming growth factor-beta (TGF-beta) regulates osteoclastogenesis and osteoclast survival, in part through the induction of osteoprotegerin (OPG), a protein known to inhibit osteoclast formation and function. To explore the molecular basis of TGF-beta regulation of OPG expression, we evaluated the effects of TGF-beta on osteoclast formation, OPG protein secretion, mRNA expression, and gene transcription. The marked inhibitory effect of TGF-beta on osteoclast differentiation was confirmed in a co-culture model utilizing murine stromal/osteoblastic BALC cells and bone marrow hematopoietic precursors. This inhibition in osteoclast differentiation was preceded by a decrease in RANKL mRNA expression (5-fold) and a reciprocal increase in OPG mRNA (6.1-fold) and protein (7.1-fold) expression in BALC cells. At the promoter/transcriptional level, TGF-beta treatment resulted in a 3-10-fold increase in reporter gene activity directed by a 5.9-kilobase fragment of the human OPG promoter in transfection assays performed in UMR106 cells. The effect of TGF-beta was mimicked by TGF-beta2 and -beta3 but not by BMP-4, suggesting a TGF-beta signal-specific effect. Deletion analysis revealed that a 183-base pair region (-372 to -190) in the promoter was required for TGF-beta responsiveness, and this region was sufficient to confer TGF-beta inducibility to a heterologous (osteocalcin) minimal promoter. Substitution mutations that disrupted the Cbfa1- and/or Smad-binding elements present in the 183-base pair region resulted in a decrease in base-line expression and in the responsiveness to TGF-beta and Cbfa1. Collectively, these studies indicate the involvement and possible interaction of Cbfa1 and Smad proteins in mediating the effects of TGF-beta on OPG transcription.

ADAMTS-1: A cellular disintegrin and metalloprotease with thrombospondin motifs is a target for parathyroid hormone in bone.

PTH stimulates bone formation in animals and humans, and the expressions of a number of genes have been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon we used differential display RT-PCR and screened for genes that are selectively expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single s.c. injection of human PTH-(1-38) (8 microg/100 g). We show that one of the messenger RNAs that is up-regulated in bone is ADAMTS-1, a new member of the ADAM (A disintegrin and metalloprotease) gene family containing thrombospondin type I motifs. ADAMTS-1 consists of multiple domains common to ADAM family of proteins, including pro-, metalloprotease-like, and disintegrin-like domains. However, unlike other ADAMs, ADAMTS-1 does not possess a transmembrane or cytoplasmic domain and is a secreted protein. Northern blot analysis confirmed that ADAMTS-1 was up-regulated in both metaphyseal (14- to 35-fold) and diaphyseal (4.2-fold) bone 1 h after PTH-(1-38) injection and returned to control levels by 24 h. We also analyzed the regulation of ADAMTS-1 in response to various PTH/PTH-related peptide (PTHrP) analogs and found that PTH-(1-31) and PTHrP-(1-34), which activate the protein kinase A (PKA) pathway, induce ADAMTS-1 expression 1 h after injection, whereas PTH-(3-34) and PTH-(7-34), which do not activate the PKA pathway, did not regulate expression. To investigate the effect of other osteotropic agents, we analyzed ADAMTS-1 expression after a single dose of PGE2 (6 mg/kg) and found that it was up-regulated 1 h after injection and returned to control levels by 6 h. In vitro ADAMTS-1 is expressed in primary osteoblasts and osteoblastic cell lines, but was not detectable in osteoclasts generated from macrophage colony-stimulating factor/receptor activator of NF-kappaB ligand/transforming growth factor-beta1-treated bone marrow cells. Treatment of UMR 106 osteosarcoma cells with PTH, PGE2, forskolin, or (Bu)2cAMP increased ADAMTS-1 expression 7-, 4-, 5-, and 5-fold, respectively. Also, in vitro treatment with 1alpha,25-dihydroxyvitamin D3 increased ADAMTS-1 expression 3-fold. Tissue distribution analysis showed that ADAMTS-1 is expressed at high levels in many tissues, including the heart, lung, liver, skeletal muscle, and kidney. Taken together, these results demonstrate that ADAMTS-1 is specifically up-regulated in bone and osteoblasts by the osteotropic agents PTH, PTHrP, and PGE2 possibly via the cAMP/PKA pathway. We speculate that the rapid and transient increase in ADAMTS-1 expression may contribute to some of the effects of PTH on bone turnover.

The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function.

Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblast-secreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE(2))), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the beta-galactosidase reporter gene (pOPG5. 9betagal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9betagal reporter construct led to 39-, 7-, and 16-fold increases in beta-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE(2) elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE(2) element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequence-specific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.

Cryptic enhancer elements in luciferase reporter vectors respond to the osteoblast-specific transcription factor Osf2/Cbfa1.

Luciferase reporter vectors are commonly used for the functional analysis of basal promoter and enhancer elements of eukaryotic genes. Randomly occurring cisacting elements in the vector sequences that can spuriously respond to various transcription factors, combined with the high sensitivity of the luciferase assay system, could make these vectors unsuitable for functional studies with certain transcription factors. Here, we provide evidence that pGL2-Basic and pGL3-Basic are transactivated by the osteoblast-specific transcription factor Cbfa1 and estrogen receptor alpha probably through randomly occurring cisacting elements in the vector sequences. Our results highlight the limitations of pGL2-Basic and pGL3-Basic vectors in promoter transactivation/repression studies. The results also emphasize the need to perform appropriate controls and test the expression levels with a particular transcription factor and promoterless luciferase reporter vector combination.

The mammalian basic helix loop helix protein HES-1 binds to and modulates the transactivating function of the runt-related factor Cbfa1.

Drosophila Runt is the founding member of a family of related transcription factors involved in the regulation of a variety of cell-differentiation events in invertebrates and vertebrates. Runt-related proteins act as both transactivators and transcriptional repressors, suggesting that context-dependent mechanisms modulate their transcriptional properties. The aim of this study was to elucidate the molecular mechanisms that contribute to the regulation of the functions of the mammalian Runt-related protein, Cbfa1. Here we provide the first demonstration that Cbfa1 (as well as the related protein, Cbfa2/AML1) physically interacts with the basic helix loop helix transcription factor, HES-1, a mammalian counterpart of the Drosophila Hairy and Enhancer of split proteins. This interaction is mediated by the carboxyl-terminal domains of Cbfa1 and HES-1, but does not require their respective tetrapeptide motifs, WRPY and WRPW. Our studies also show that HES-1 can antagonize the binding of Cbfa1 to mammalian transcriptional corepressors of the Groucho family. Moreover, HES-1 can potentiate Cbfa1-mediated transactivation in transfected cells. Taken together, these findings implicate HES-1 in the transcriptional functions of Cbfa1 and suggest that the concerted activities of Groucho and HES proteins modulate the functions of mammalian Runt-related proteins.

Dynamic regulation of RGS2 in bone: potential new insights into parathyroid hormone signaling mechanisms.

The initial steps involved in mediating the transduction of PTH signal via its G protein-coupled receptors are well understood and occur through the activation of cAMP and phospholipase C pathways. However, the cellular and molecular mechanisms for subsequent receptor desensitization are less well understood. Recently, a new family of GTPase activating proteins known as regulators of G protein signaling (RGS), has been implicated in desensitization of several G protein-coupled ligand-induced processes. At present, it is not known whether any of the RGS proteins play a role in PTH signaling. Using the differential display method, we screened for genes that are selectively expressed after a single s.c. injection of human PTH (1-38) (8 microg/100 g) in osteoblast-enriched femoral metaphyseal spongiosa of young male rats (3-4 weeks old). We found and cloned one full-length complementary DNA that encodes a 211-amino acid RGS protein and shares 97% sequence identity with mouse and human RGS2. Based on sequence similarity, we have designated this clone as rat RGS2. Northern blot analysis confirmed that the expression of RGS2 messenger RNA (mRNA) is rapidly and transiently increased by human PTH (1-38) in both metaphyseal (4-to 5-fold) and diaphyseal (2- to 3-fold) bone, as well as in cultured osteoblast cultures (2- to 37-fold). In vitro, forskolin and dibutyryl cAMP similarly elevated RGS2 mRNA. In vivo, PTH analog (1-31) [which stimulates intracellular cAMP accumulation, PTHrP (1-34), and prostaglandin E2] induced RGS2 mRNA expression; whereas PTH analogs (3-34) and (7-34), which do not stimulate cAMP production, had no effect on expression. In tissue distribution analysis, RGS2 is widely expressed and was detected in all tissues examined (heart, spleen, liver, skeletal muscle, kidney, and testis), with significant expression in two nonclassical PTH-sensitive tissues: the brain, and the heart. After PTH injection, RGS2 mRNA expression was induced in rat bone but not in any of the other tissues examined. These findings demonstrate that RGS2 is regulated by PTH, prostaglandin E2, and PTHrP and that regulation by PTH in bone occurs via the cAMP pathway. Additionally, these results suggest the exciting possibility that increased RGS2 expression in osteoblasts may be one of the early events influencing PTH signaling.

CBFA1 mutation analysis and functional correlation with phenotypic variability in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a dominantly inherited skeletal dysplasia caused by mutations in the osteoblast-specific transcription factor CBFA1. To correlate CBFA1 mutations in different functional domains with the CCD clinical spectrum, we studied 26 independent cases of CCD and a total of 16 new mutations were identified in 17 families. The majority of mutations were de novo missense mutations that affected conserved residues in the runt domain and completely abolished both DNA binding and transactivation of a reporter gene. These, and mutations which result in premature termination in the runt domain, produced a classic CCD phenotype by abolishing transactivation of the mutant protein with consequent haploinsufficiency. We further identified three putative hypomorphic mutations (R391X, T200A and 90insC) which result in a clinical spectrum including classic and mild CCD, as well as an isolated dental phenotype characterized by delayed eruption of permanent teeth. Functional studies show that two of the three mutations were hypomorphic in nature and two were associated with significant intrafamilial variable expressivity, including isolated dental anomalies without the skeletal features of CCD. Together these data show that variable loss of function due to alterations in the runt and PST domains of CBFA1 may give rise to clinical variability, including classic CCD, mild CCD and isolated primary dental anomalies.

Disseminated nocardiosis in a bone marrow transplant recipient with chronic GVHD.

We describe a case of disseminated nocardiosis in a 53-year-old male allogeneic marrow recipient with chronic GVHD, 15 years post BMT. Six months prior to admission he was treated for recurrent chronic GVHD with corticosteroids with a good response. He deteriorated subsequently while still on steroids requiring admission for fever, anorexia, weight loss, productive cough and progressive dyspnoea. On admission he had multiple nodular lesions on chest roentgenogram and subsequently grew Nocardia farcinica in blood culture. N. farcinica is rare post BMT, has a high mortality, is resistant to various antibiotics and needs prolonged antimicrobial therapy. We report the successful management of our patient with single agent trimethoprim-sulphamethoxazole.

Two domains unique to osteoblast-specific transcription factor Osf2/Cbfa1 contribute to its transactivation function and its inability to heterodimerize with Cbfbeta.

Osf2/Cbfa1, hereafter called Osf2, is a member of the Runt-related family of transcription factors that plays a critical role during osteoblast differentiation. Like all Runt-related proteins, it contains a runt domain, which is the DNA-binding domain, and a C-terminal proline-serine-threonine-rich (PST) domain thought to be the transcription activation domain. Additionally, Osf2 has two amino-terminal domains distinct from any other Runt-related protein. To understand the mechanisms of osteoblast gene regulation by Osf2, we performed an extensive structure-function analysis. After defining a short Myc-related nuclear localization signal, a deletion analysis revealed the existence of three transcription activation domains and one repression domain. AD1 (for activation domain 1) comprises the first 19 amino acids of the molecule, which form the first domain unique to Osf2, AD2 is formed by the glutamine-alanine (QA) domain, the second domain unique to Osf2, and AD3 is located in the N-terminal half of the PST domain and also contains sequences unique to Osf2. The transcription repression domain comprises the C-terminal 154 amino acids of Osf2. DNA-binding, domain-swapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbfbeta, a known partner of Runt-related proteins, whereas a deletion mutant of Osf2 containing only the runt and PST domains does. The QA domain appears to be responsible for preventing this heterodimerization. Thus, our results uncover the unique functional organization of Osf2 by identifying functional domains not shared with other Runt-related proteins that largely control its transactivation and heterodimerization abilities.

Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder characterized by hypoplastic or absent clavicles, large fontanelles, dental anomalies and delayed skeletal development. The phenotype is suggestive of a generalized defect in ossification and is one of the most common skeletal dysplasias not associated with disproportionate stature. To date, no genetic determinants of ossification have been identified. CCD has been mapped to chromosome 6p21, where CBFA1, a gene encoding OSF2/CBFA1, a transcriptional activator of osteoblast differentiation, has been localized. Here, we describe two de novo missense mutations, Met175Arg and Ser191Asn, in the OSF2/CBFA1 gene in two patients with CCD. These two mutations result in substitution of highly conserved amino acids in the DNA-binding domain. DNA-binding studies with the mutant polypeptides show that these amino acid substitutions abolish the DNA-binding ability of OSF2/CBFA1 to its known target sequence. Concurrent studies show that heterozygous nonsense mutations in OSF2/CBFA1 also result in CCD, while mice homozygous for the osf2/cbfa1 mull allele exhibit a more severe lethal phenotype. Thus, these results together suggest that CCD is produced by haploinsufficiency of OSF2/CBFA1 and provide direct genetic evidence that the phenotype is secondary to an alteration of osteoblast differentiation.

Temperature-sensitive mutations in the protein kinase-1 (pk-1) gene of the Autographa californica nuclear polyhedrosis virus that block very late gene expression.

We have developed a genetic screen for temperature-sensitive mutations in the very late transcription apparatus of the Autographa californica nuclear polyhedrosis virus. This method starts with the BacPAKS virus, which has the Escherichia coli lacZ gene under the control of the very late polyhedrin promoter. The desired mutants are temperature-sensitive for beta-galactosidase production and can be complemented by wild-type virus, which lacks the lacZ gene. Two mutants created by nitrosoguanidine mutagenesis and identified by this screen, and one mutant identified by another screen, have been mapped by marker rescue to the viral protein kinase 1 gene (pk-1). The protein kinase genes of these three mutant viruses have been sequenced, revealing the same point mutation in two of them and a different point mutation in the other. In each case, a single amino acid is changed: In two mutants, XF4 and XF5, Asp 92 is changed to Asn; in the other mutant, KT800, Thr 204 is changed to lle. Northern blotting of RNA made in cells infected by these three mutant viruses has shown that the accumulation of very late transcripts (lacZ and p10) is temperature-sensitive, but that accumulation of at least one late transcript (vp39) is not temperature-sensitive. Nuclear run-on transcription assays with two of the mutants indicate that very late transcription is somewhat temperature-sensitive, although this defect is not as pronounced as the temperature-sensitivity detected by Northern blotting. Transcription of at least one late gene (vp39) is not temperature-sensitive in cells infected by these two mutants. Thus, it appears that the viral protein kinase-1 is involved in very late gene expression. Some of this effect is at the transcription level, but some may also be exerted at the posttranscription level.