Recurrent central odontogenic fibroma in a patient with nevoid basal cell carcinoma syndrome: case report and in vitro analysis.

OBJECTIVE: Central odontogenic fibromas (COF) are rare, benign tumors derived from dental mesenchymal tissue that may occur in the maxilla or mandible. This report describes primary and recurrent COF in the mandible of a patient with nevoid basal cell carcinoma syndrome (NBCCS). STUDY DESIGN: A 36-year-old African American male presented with a COF and its recurrence 17 months later. Tissue pieces were obtained from both occurrences with IRB-approved signed consent. Collected tissue pieces were dissected; one portion was formalin-fixed and paraffin-embedded, and the other was cultured for the isolation of cell populations from the primary (COdF-1) and recurrent (COdF-1a) tumors. Quantification real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, and DNA sequencing were used for gene and protein analysis of the primary tumor and cell populations. RESULTS: Histopathologic analysis of the tumor showed sparse odontogenic epithelial cords in fibrous connective tissue, and qRT-PCR analysis of tumor and cell populations (COdF-1 and COdF-1a) detected VIM, CK14, CD34, CD99 and ALPL mRNA expression. Protein expression was confirmed by immunohistochemistry. CD34 expression in primary tissues was higher than in tumor cells due to tumor vascularization. DNA sequencing indicated the patient had PTCH1 mutations. CONCLUSIONS: Histopathology, mRNA, and protein expression indicate the rare occurrence of COF in a patient with mutated PTCH1 gene and NBCCS.

Bioefficacy evaluation of ferrocenyl chalcones against Meloidogyne incognita and Sclerotium rolfsii infestation in tomato.

The present study reports, bioefficacy evaluation of effective compounds against Meloidogyne incognita and Sclerotium rolfsii in pot cultured tomato. The identified five most effective compounds, i.e. (2E)-1-(4-Methylphenyl)-3-ferrocenyl-prop-2-en-1-one (6g), (2E)-1-(4-Methoxyphenyl)-3-ferrocenyl-prop-2-en-1-one (6h), (2E)-1-(3-Bromophenyl)-3-ferrocenyl-prop-2-en-1-one (6j), (2E)-1-(2,4-Dichlorophenyl)-3-ferrocenyl-prop-2-en-1-one (6k) and (2E)-1-(3,5-Dichloro-2-hydroxyphenyl)-3-ferrocenyl-prop-2-en-1-one (6p) along with Carbofuran 3G as positive control were tested at 20, 40 and 80 ppm by soil drenching and root dipping methods. The study revealed that all plant growth parameters were positively influenced by these compounds. The presence of an electron releasing group positively influenced the efficacy, and the activity was highest in compounds 6g and 6h at 80 ppm. Based on in vitro results against S. rolfsii, (2E)-1-Ferrocenyl-3-(4-bromophenyl)-prop-2-en-1-one (3b), (2E)-1-Ferrocenyl-3-(2,6-dichlorophenyl)-prop-2-en-1-one (3o) and (2E)-1-(5-Chloro-2-hydroxyphenyl)-3-ferrocenyl-prop-2-en-1-one (6o) along with Tebuconazole 25.9% EC and Hexaconazole 5% SC as positive control were evaluated. The shoot length was found to be highest (24.50 cm) in plants treated with 3b followed by 3o and 6o at 1000 ppm. The percent disease incidence was significantly decreased as compared to control. The percent disease incidence was found to be minimum in plants treated with 3b at 1000 ppm. However, root dipping was not as effective as soil drenching. Therefore, ferrocenyl chalcone derivatives proved to be of great fungicidal and nematicidal potential opening new opportunities for expanding their effectiveness as new pest control agents.

Evaluation of changes in corneal endothelium in chronic kidney disease.

Purpose: Chronic kidney disease (CKD) is an emerging health problem worldwide. In CKD corneal endothelial changes also occur probably due to accumulation of inflammatory cytokines and increased multiple toxic products. The aim of this study was to analyze the effect of CKD on corneal endothelium and correlate the findings with severity of disease with help of noninvasive technique. Methods: The study comprised 75 eyes of 75 cases divided into three groups with group A comprising of CKD cases on dialysis, group B of nondialysis CKD cases, and group C of controls. Each group had 25 cases each of either sex and between 15-80 age groups. All patients were investigated for blood urea, serum creatinine, and blood sugar and underwent complete ophthalmic examination of both eyes along with wide-field specular microscopy examination. Results: The majority of patients (33.3%) belonged to age range of 61-70 years with male predominance and the most common cause of CKD was found to be diabetes with 17 (34%) cases. We found normal corneal endothelial cell density (ECD) with the mean ECD of 2364.52 ± 397.72 mm2 in the dialysis group, 2467.8 ± 352.88 mm2 in nondialysis group, and 2521.68 ± 250.26 mm2 in the control group of patients. However, we found significant increase in coefficient of variation (CV) with 36 ± 5.8% in dialysis group, 37 ± 4.5% in nondialysis group and 32 ± 0.8% in controls (P = 0.001) and decreased hexagonality (Hx) with 47 ± 7.3% in dialysis group, 46 ± 4.7% in nondialysis group and 51 ± 6.7% in the controls (P = 0.031). This showed increased tendency of pleomorphism and polymegathism in corneal endothelial cells in CKD cases. No correlation was found between blood urea or serum creatinine levels with endothelial parameters in any group. Conclusion: CKD causes morphological changes like polymegathism and pleomorphism in corneal endothelium and hence these cases are more vulnerable and special care should be taken before any intraocular surgical procedure.

Microwave assisted synthesis, characterization and biological activities of ferrocenyl chalcones and their QSAR analysis: Part II.

A series of ferrocenyl chalcones using acetylferrocene, with ferrocenyl group at the keto carbonyl group, and different aldehydes were synthesized and their bioefficacy evaluation was done against Sclerotium rolfsii, Alternaria solani and Meloidogyne incognita. In continuation of our quest for potent crop protection products, in the present study, a series of 18 substituted ferrocenyl chalcones were synthesized in which ferrocenyl group was attached to the aldehyde moiety, using ferrocenecarboxyaldehyde and different acetophenones by microwave method (MM) and conventional method (CM) [cf: MM 1 to 5 min; CM 12-40 h] and characterized by various techniques viz. IR, LC-HRMS, 1H-NMR and 13C-NMR. In vitro fungicidal activity showed that compound, (2E)-1-(5-Chloro-2-hydroxyphenyl)-3-ferrocenyl-prop-2-en-1-one (34) (ED50 = 21.50 mg L-1) was found to be most active against S. rolfsii and compound, (2E)-1-(4-Bromophenyl)-3-ferrocenyl-prop-2-en-1-one (21) (ED50 = 31.14 mg L-1) showed highest activity against A. solani. As regards nematicidal activity, compound (2E)-1-(3-Bromophenyl)-3-ferrocenyl-prop-2-en-1-one (29) was more potent with LC50 values of 11.95, 8.07 and 4.34 mg L-1 at 24, 48 and 72 h, respectively. QSAR study revealed that MLR for S. rolfsii (r 2 = 0.9834, q 2= 0.8975) and A. solani (r 2 = 0.9807, q 2= 0.8713) and PLS for M. incognita (r 2 = 0.9023, q 2= 0.7818) were the best models.

Microwave Assisted Synthesis, Characterization and Biological Activities of Ferrocenyl Chalcones and Their QSAR Analysis.

A new microwave method (MM) has been developed for the synthesis of a series of 16 substituted ferrocenyl chalcones using acetylferrocene (1) with different aldehydes (2a-2p) and comparing it with conventional method (CM). The synthesized compounds were characterized by various spectroscopic techniques viz IR, HR-MS, 1H NMR, and 13C NMR. The time required for completion of reaction in MM varied from 1 to 5 min as compared to CM which required 10-40 h. All the synthesized compounds were screened for antifungal activity against Sclerotium rolfsii and Alternaria solani. In vitro fungicidal activity revealed that compound 3o (ED50 = 23.24 mg L-1) was found to be most active against S. rolfsii. But in case of A. solani, compound 3c (ED50 = 29.9 mg L-1) showed highest activity. The nematicidal activity revealed that the compound 3b was more potent with LC50 values of 10.67, 7.30, and 4.55 ppm at 24, 48, and 72 h, respectively. 2D-Quantitative Structural Activity Relationship (2D-QSAR) analysis of these ferrocenyl chalcones was carried out by developing three different models namely Partial Least Squares (PLS, Model 1), Multiple Linear Regression (MLR, Model 2) and Principal Component Regression (PCR, Model 3). Statistical significance and predictive ability of these models were assessed by internal and external validation and also verified by leave one-out cross-validation. QSAR study revealed that MLR for S. rolfsii (r 2 = 0.999, q 2 = 0.996), PLS for A. solani (r 2 = 0.934, q 2 = 0.749) and PCR for M. incognita (r 2 = 0.878, q 2 = 0.772) were the best model. The physico-chemical parameters were calculated using VLife MDS 4.6 software. QSAR study could be employed for structure optimization to achieve better activity.

Quorum sensing pathways in Gram-positive and -negative bacteria: potential of their interruption in abating drug resistance.

Quorum sensing (QS) is an inter-cell communication between bacterial populations through release of tiny diffusible compounds as signalling agents, called auto-inducers, abetting bacteria to track population density. QS allows bacterial population to perform collectively in coordination to wide phenotypes like alterations in expression of virulence genes to achieve advancement over their competitors, drug resistance and biofilm formation. Several classes of autoinducers have been described that are involved in bacterial virulence. This review gives an insight into the multitudinous QS systems in Gram-positive and Gram-negative bacteria to explore their role in microbial physiology and pathogenesis. Bacterial resistance to antibiotics has clinically become a super challenge. Strategies to interrupt QS pathways by natural and synthetic QS inhibitors or quorum quenchers or analogs provide a potential treatment. We highlight the advancements in discovery of promising new targets for development of next generation antimicrobials to control infections caused by multidrug resistant bacterial pathogens.

1H, 15N, and 13C chemical shift assignments of the regulatory domain of human calcineurin.

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM-CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete 13C, 15N and 1H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of 13C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A 15N-resolved TOCSY experiment has been used to assign Hα and Hß chemical shifts.

Purity Evaluation of Curcuminoids in the Turmeric Extract Obtained by Accelerated Solvent Extraction.

Curcuminoids, the active principle of Curcuma longa L, is one of the most researched subjects worldwide for its broad-spectrum biological activities. Being traditionally known for their anticancer properties and issues related to bioavailability, the curcuminoids, including diferuloylmethane (curcumin), have gained special attention. Thus, the current study focused on the purity profiling of curcuminoids when extracted by accelerated solvent extraction, which was run with turmeric rhizome powder (20 g) at 1500 psi and at 50°C, with a static time of 10 min and with three cycles. The performance of ethanol, ethyl acetate, and acetone as extraction solvents was comparatively evaluated. Once extracted, the individual curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) were purified by column chromatography, followed by preparative TLC, and the compounds were characterized by spectroscopic and chromatographic techniques. The HPLC method was standardized by using a gradient mobile phase of water and acetonitrile containing 0.1% formic acid. The LODs were calculated as 0.27, 0.33, and 0.42 µg/mL for curcumin, demethoxycurcumin, and bisdemethoxycurcumin, respectively. Accuracy (relative percentage error) and precision RSD values of the developed HPLC method were below 5%. The intraday accuracy ranged between -0.9 and -3.63%. The physical yield was the highest in ethanol (8.4%) extraction, followed by ethyl acetate (7.4%) and acetone (6.6%). Maximum purity was recorded in acetone (46.2%), followed by ethanol (43.4%) and ethyl acetate (38.8%), with no significant differences across the individual curcuminoids. This research will be useful for future applications related to the extraction of curcuminoids at a commercial level and to their profiling in food matrixes.

An insight into fusion technology aiding efficient recombinant protein production for functional proteomics.

Advancements in peptide fusion technologies to maximize the protein production has taken a big leap to fulfill the demands of post-genomics era targeting elucidation of structure/function of the proteome and its therapeutic applications, by over-expression in heterologous expression systems. Despite being most preferred protein expression system armed with variety of cardinal fusion tags, expression of the functionally active recombinant protein in E. coli remains plagued. The present review critically analyses the aptness of well-characterized fusion tags utilized for over-expression of recombinant proteins with improved solubility and their compatibility with downstream purification procedures. The combinatorial tandem affinity strategies have shown to provide more versatile options. Solubility decreasing fusion tags have proved to facilitate the overproduction of antimicrobial peptides. Efficient removal of fusion tags prior to final usage is of utmost importance and has been summarized discussing the efficiency of various enzymatic and chemical methods of tag removal. Unfortunately, no single fusion tag works as a magic bullet to completely fulfill the requirements of protein expression and purification in active form. The information provided might help in selection and development of a successful protocol for efficient recombinant protein production for functional proteomics.

Understanding Protein Structure Deformation on the Surface of Gold Nanoparticles of Varying Size.

Gold nanoparticles (AuNPs) have been of recent interest due to their unique optical properties and their biocompatibility. Biomolecules spontaneously adsorb to their surface, a trait that could potentially be exploited for drug targeting. Currently, it is unclear whether protein-AuNP interactions at the nanoparticle surface are dependent on nanoparticle size. In this work, we investigate whether varying surface curvature can induce protein unfolding and multilayer binding in citrate-coated AuNPs of various sizes. A recently developed NMR-based approach was utilized to determine the adsorption capacity, and protein NMR spectra were compared to determine whether nanoparticle size influences protein interactions at the surface. In addition, transmission electron microscopy (TEM) and dynamic light scattering (DLS) were employed to corroborate the NMR studies. Over a broad range of AuNP sizes (14-86 nm), we show that adsorption capacity can be predicted by assuming that proteins are compact and globular on the nanoparticle surface. Additionally, roughly one layer of proteins is adsorbed regardless of AuNP size. Our results hold for two proteins of significantly different sizes, GB3 (6 kDa) and bovine carbonic anhydrase (BCA, 29 kDa). However, the unstable drkN SH3 domain (ΔG0 ≈ 0, 7 kDa) does not appear to follow the same trend seen for stable, globular proteins. This observation suggests that unstable proteins can deform significantly when bound to AuNP surfaces. Taken together, the results of this work can be used to improve our knowledge of the mechanism of protein-AuNP interactions to optimize their use in the biomedical field.

Isolation, in silico characterization, localization and expression analysis of abiotic stress-responsive rice G-protein ß subunit (RGB1).

Heterotrimeric G-proteins constitute the classical signaling paradigm along with their cognate G-protein coupled receptors (GPCRs) and appropriate downstream effectors. G-protein complex is composed of highly conserved Gα, Gß, and Gγ subunits. In the present study, we have characterized the cis-regulatory elements of the promoter, signature motifs, transcript profile in response to abiotic stresses, and sub-cellular localization of G-protein ß subunit RGB1(I) from Indica rice. The RGB1(I) promoter sequence has various stress-related cis-regulatory elements suggesting its role in abiotic stress signaling. Presence of six WD-40 repeat signature motifs in RGB1(I) suggest its role in exchange of GDP by GTP in Gα subunit and receptor recognition. Presence of multiple N-myristoylation consensus sites in RGB1(I) protein sequence, which is necessary for membrane localization of protein, confirms the association of RGB1(I) in plasma membrane. Extrinsic association of RGB1(I) with plasma membrane seems essential for its role in regulation of signaling pathways and adaptation to high salt stress. We report the sub-cellular localization of RGB1(I) in plasma membrane, cytosol and nucleus. The localization of RGB1(I) in nucleus supports its possible interaction with transcription factors regulating the expression of salt stress responsive genes. The RGB1(I) transcript was upregulated under KCl, cold, dehydration and micronutrient (Mn (2+) and Zn (2+)) stress. However, transcript variation under elevated temperature, ABA, NaCl, and toxic heavy metals (viz. arsenite, arsenate, cadmium and lead) was not encouraging. These evidences indicate an active and significant role of RGB1(I) in the regulation of abiotic stresses in rice and propound its possible exploitation in the development of abiotic stress tolerance in crops.

Greater Skeletal Gains in Ovary Intact Rats at Maturity Are Achieved by Supplementing a Standardized Extract of Butea monosperma Stem Bark that Confers Better Bone Conserving Effect following Ovariectomy and Concurrent Treatment Withdrawal.

With a longitudinally designed study, we tested whether an acetone soluble fraction (ASF) from the stem bark of Butea monosperma resulted in maximizing bone gain in rats during growth and maturation and thus protected against osteopenia following ovariectomy (OVx) with concomitant treatment withdrawal. Female rats at weaning were given ASF (100 mg/kg/d) or vehicle for 12 weeks, and baseline skeletal parameters (micro-CT) and total plasma antioxidant status (TAS) were measured. At this stage, one group was OVx and the other group was sham operated. Vehicle group (untreated) after OVx was given E2 or continued with vehicle (OVx control). ASF group after OVx was given vehicle (ASF withdrawn, ASFW). After another 12 weeks, all groups were killed and various skeletal parameters were determined. ASF resulted in substantially better skeletal parameters and higher plasma TAS over control at maturity. Rats treated with ASF before OVx had reduced rates of bone loss compared to OVx control. Twelve weeks after OVx, the ASFW group exhibited better trabecular microarchitectural preservation, bone turnover profiles, increased cortical deposition, and biomechanical strength over the OVx control, and the effects were comparable to OVx + E2 group. ASF supplementation during skeletal growth could maximize bone accrual and could confer increased resistance to post-OVx osteopenia despite treatment withdrawal.

Tacrolimus-induced severe cholestasis complicating renal transplantation.

Tacrolimus, a calcineurin inhibitor, is a potent immunosuppressive agent used by a majority of transplanters across the globe. Its adverse effects include nephrotoxicity, neurotoxicity, new onset diabetes after transplant, gastro-intestinal toxicity, hepatotoxicity, and thrombotic microangiopathy. Tacrolimus-induced hepatotoxicity is a very uncommon side effect. We report a case of tacrolimus-induced hepatotoxicity in the form of cholestatic hepatitis a renal transplant recipient. Hepatotoxicity did not decrease after dose reduction; however, normalization of liver enzymes occurred after discontinuing tacrolimus.

Rice heterotrimeric G-protein alpha subunit (RGA1): in silico analysis of the gene and promoter and its upregulation under abiotic stress.

Heterotrimeric G-protein complexes (Gα, Gß and Gγ) operate at the apex of diverse signal transduction systems along with their cognate transmembrane G-protein coupled receptors (GPCRs) and appropriate downstream effectors in the plant. Rice Gα in response to stress has not been well studied. Here, we report the in silico analysis of Gα subunit from Oryza sativa cv. Indica group Swarna [RGA1(I), accession number HQ634688], its promoter and its transcript upregulation in response to abiotic stresses. Genomic sequence of RGA1(I) contains thirteen exonic and twelve intronic segments. Phylogenetic analysis of RGA1(I) demonstrated high homology with Sorghum and maize and is distantly related to barley and wheat. Promoter sequence analysis of RGA1(I) confirms the presence of stress-related cis-regulatory elements viz. ABA, MeJAE, ARE, GT-1 boxes and LTR suggesting its active and possible independent roles in abiotic stress signalling. Expasy PROSITE database of protein families and domains revealed important motifs, patterns and biologically significant sites in RGA1(I). Three dimensional structure of RGA1(I) protein predicted by I-TASSER server and its stereochemical qualities were validated by PROCHECK and QMEAN server indicating the acceptability of the predicted model. The transcript profiling of RGA1(I) showed upregulation following NaCl, cold and drought stress. Under elevated temperature, its transcript was down regulated. Heavy metal(loid)s stress showed rhythmic and strong upregulation. It showed a rhythmic response in ABA stress. These findings provide a critical evidence for its active role in regulation of abiotic stresses in rice. These findings suggest its possible exploitation in the development of abiotic stress tolerance in crops.

Localization of rabies virus glycoprotein into the endoplasmic reticulum produces immunoprotective antigen.

Rabies virus surface glycoprotein (rabies G-protein) with (G+RS) and without (G-RS) endoplasmic reticulum retrieval signal was expressed and characterized in tobacco plants. Transgenically expressed rabies G-protein was estimated at 0.015-0.38 % of total leaf protein. The relative migration of the rabies G-protein on SDS-PAGE was at the position, as anticipated for the viral coat protein (~66 kDa). Immunolocalization by confocal microscopy established that immunoprotective G+RS expressed in tobacco was primarily confined to ER. G+RS showed binding to Con A lectin and was susceptible to N-glycosidase F activity similar to native rabies G-protein. However, the G-RS transgenically expressed in tobacco leaves was glycosylated differently and was resitant to N-glycosidase F. Immunological studies and Rapid Fluorescent Foci Inhibition Test (RFFIT) showed that G+RS was immunogenic and immunoprotective, whereas G-RS was moderately immunogenic but non-protective against live virus challenge. Hence, plants can express the antigenic component of rabies virus with suitable glycosylation, which is important to give protection against rabies virus infection.

Anti-ulcer constituents of Annona squamosa twigs.

Phytochemical investigation of Annona squamosa twigs, resulted in isolation and identification of twelve known (1-12) compounds among them one 1-(4-ß-D-glucopyranosyloxyphenyl)-2-(ß-D-glucopyranosyloxy)-ethane (11) is synthetically known but first time isolated from natural sources. Their structures were elucidated using 1D and 2D NMR spectroscopic analysis. The isolated compounds (2-8, 11) were evaluated for H(+) K(+)-ATPase activity. Three of these compounds (+)-O-methylarmepavine (2), N-methylcorydaldine (3), isocorydine (6) showed promising anti-secretory activity. Activity of these compounds, comparable to the standard drug omeprazole is novel to our finding. Moreover, there is no information accessible regarding the pharmacological effect of A. squamosa on the gastrointestinal system. This study is the first of its kind to show the significant anti-ulcer effect of A. squamosa. The present study aimed to evaluate the gastroprotective effect of A. squamosa (AS) and to identify its active constituents. Anti-ulcer activity was evaluated against cold restraint (CRU), pyloric ligation (PL), aspirin (ASP), alcohol (AL) induced gastric ulcer and histamine (HA) induced duodenal ulcer model and further confirmed through in vitro assay of H(+) K(+)-ATPase activity and plasma gastrin level. AS and its chloroform and hexane fraction attenuated ulcer formation in CRU, PL, HA model and displayed anti-secretory activity in vivo through reduced free, total acidity and pepsin in PL, confirmed by in vitro inhibition of H(+) K(+)-ATPase activity with corresponding decrease in plasma gastrin level. Cytoprotection of AS was apparent with protection in AL, ASP models and enhanced mucin level in PL.

Synthetic analogs of daidzein, having more potent osteoblast stimulating effect.

A series of didzein derivatives were synthesized and assessed for stimulation of osteoblast differentiation using primary cultures of rat calvarial osteoblasts. Data suggested that three synthetic analogs, 1c, 3a and 3c were several folds more potent than daidzein in stimulating differentiation and mineralization of osteoblasts. Further, these three compounds did not show any estrogen agonistic activity, however had mild estrogen antagonistic effect. Out of the three compounds, 3c was found to maximally increase the mineralization of bone marrow osteoprogenitor cells. Compound 3c also robustly increased the mRNA levels of osteogenic genes including bone morphogenetic protein-2 and osteocalcin in osteoblasts. Unlike daidzein, 3c did not inhibit osteoclastogenesis. Collectively, we demonstrate osteogenic activity of daidzein analogs at significantly lower concentrations than daidzein.

Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats.

Dietary soy isoflavones including genistein and daidzein have been shown to have favorable effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of cladrin and formononetin, two structurally related methoxydaidzeins found in soy food and other natural sources. Cladrin, at as low as 10 nM, maximally stimulated both osteoblast proliferation and differentiation by activating MEK-Erk pathway. On the other hand, formononetin maximally stimulated osteoblast differentiation at 100 nM that involved p38 MAPK pathway but had no effect on osteoblast proliferation. Unlike daidzein, these two compounds neither activated estrogen receptor in osteoblast nor had any effect on osteoclast differentiation. Daily oral administration of each of these compounds at 10.0 mg kg(-1) day(-1) dose to recently weaned female Sprague-Dawley rats for 30 consecutive days, increased bone mineral density at various anatomic positions studied. By dynamic histomorphometry of bone, we observed that rats treated with cladrin exhibited increased mineral apposition and bone formation rates compared with control, while formononetin had no effect. Cladrin had much better plasma bioavailability compared with formononetin. None of these compounds exhibited estrogen agonistic effect in uteri. Our data suggest that cladrin is more potent among the two in promoting parameters of peak bone mass achievement, which could be attributed to its stimulatory effect on osteoblast proliferation and better bioavailability. To the best of our knowledge, this is the first attempt to elucidate structure-activity relationship between the methoxylated forms of daidzein and their osteogenic effects.