Rice husk ash based growing media impact on cucumber and melon growth and quality.

Rice husk, an agricultural waste from the rice industry, can cause serious environmental pollution if not properly managed. However, rice husk ash (RHA) has been found to have many positive properties, making it a potential replacement for non-renewable peat in soilless planting. Thus, this study investigated the impact of a RHA composite substrate on the growth, photosynthetic parameters, and fruit quality of cucumber (Yuyi longxiang variety) and melon (Yutian yangjiaomi variety). The RHA, peat, vermiculite, and perlite were blended in varying proportions, with the conventional seedling substrate (peat:vermiculite:perlite = 1:1:1 volume ratio) serving as the control (CK). All plants were cultivated in barrels filled with 10L of the mixed substrates. The results from this study found that RHA 40 (RHA:peat:vermiculite:perlite = 4:4:1:1 volume ratio) significantly enhanced substrate ventilation and positively influenced the stem diameter, root activity, seedling index, chlorophyll content, net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) of cucumber and melon plants. Additionally, plant planted using RHA 40, the individual fruit weight of cucumber and melon found to increase by 34.62% and 21.67%, respectively, as compared to the control. Aside from that, both cucumber and melon fruits had significantly higher sucrose, total soluble sugar, vitamin C, and soluble protein levels. This subsequently improved the activity of sucrose synthase and sucrose phosphate synthase in both cucumber and melon. In conclusion, the RHA 40 found to best promote cucumber and melon plant growth, increase plant leaf photosynthesis, and improve cucumber and melon fruit quality, making it a suitable substrate formula for cucumber and melon cultivation in place of peat.

Moderated mediation role of ethnicity on natural skin care products purchasing intention model among multicultural consumers.

The trends for sustainable lifestyle and marketing motivated natural product consumption, such as natural skin care products (NSCPs). Different personal, environmental, and sociocultural factors influence purchase intention (PI) for NSCPs. However, there is a lack of evidence on the role of consumers' ethnicity in the PI model. The present study investigated the moderated mediation role of ethnicity in the relationship between related factors, including environmental concern, subjective norms, health factor, Halal certificate, packaging design, past experience factor, price factor, and PI mediated by personal attitude. A web-based survey was utilized to capture quantitative data from a random sample of 330 multicultural consumer group participants. The results of the study indicated that consumers' ethnicity substantially moderated the mediation effect of personal attitude in the relationships between subjective norms, health factor, Halal certificate, packaging design, past experience factor, price factor, and PI in the model. The findings contributed to understanding of the factors that influenced the PI of consumers from diverse sociocultural contexts in the market for natural products. It contributed directly to natural product marketing and industry.

In silico identification and in vitro assessment of a potential anti-breast cancer activity of antimicrobial peptide retrieved from the ATMP1 Anabas testudineus fish peptide.

A previous study has shown that synthetic antimicrobial peptides (AMPs) derived from Anabas testudineus (ATMP1) could in-vitro inhibit the progression of breast cancer cell lines. In this study, we are interested in studying altered versions of previous synthetic AMPs to gain some insight into the peptides functions. The AMPs were altered and subjected to bioinformatics prediction using four databases (ADP3, CAMP-R3, AMPfun, and ANTICP) to select the highest anticancer activity. The bioinformatics in silico analysis led to the selection of two AMPs, which are ATMP5 (THPPTTTTTTTTTTTYTAAPATTT) and ATMP6 (THPPTTTTTTTTTTTTTAAPARTT). The in silico analysis predicted that ATMP5 and ATMP6 have anticancer activity and lead to cell death. The ATMP5 and ATMP6 were submitted to deep learning databases (ToxIBTL and ToxinPred2) to predict the toxicity of the peptides and to (AllerTOP & AllergenFP) check the allergenicity. The results of databases indicated that AMPs are non-toxic to normal human cells and allergic to human immunoglobulin. The bioinformatics findings led to select the highest active peptide ATMP5, which was synthesised and applied for in-vitro experiments using cytotoxicity assay MTT Assay, apoptosis detection using the Annexin V FTIC-A assay, and gene expression using Apoptosis PCR Array to evaluate the AMP's anticancer activity. The antimicrobial activity is approved by the disc diffusion method. The in-vitro experiments analysis showed that ATMP5 had the activity to inhibit the growth of the breast cancer cell line (MDA-MB-231) after 48 h and managed to arrest the cell cycle of the MDA-MB-231, apoptosis induction, and overexpression of the p53 by interaction with the related apoptotic genes. This research opened up new opportunities for developing potential and selective anticancer agents relying on antimicrobial peptide properties.

Screening of high-risk deleterious missense variations in the CYP1B1 gene implicated in the pathogenesis of primary congenital glaucoma: A comprehensive in silico approach.

Background: Primary congenital glaucoma (PCG) is the most common subtype of glaucoma caused by defects in the cytochrome P450 1B1 (CYP1B1) gene. It is developing among infants in more than 80% of cases who exhibit impairments in the anterior chamber angle and the trabecular meshwork. Thus, a comprehensive in silico approach was performed to evaluate the effect of high-risk deleterious missense variations in the CYP1B1 gene. Material and methods: All the information for CYP1B1 missense variants was retrieved from the dbSNP database. Seven different tools, namely: SIFT, PolyPhen-2, PROVEAN, SNAP2, PANTHER, PhD-SNP, and Predict-SNP, were used for functional annotation, and two packages, which were I-Mutant 2.0 and MUpro, were used to predict the effect of the variants on protein stability. A phylogenetic conservation analysis using deleterious variants was performed by the ConSurf server. The 3D structures of the wild-type and mutants were generated using the I-TASSER tool, and a 50 ns molecular dynamic simulation (MDS) was executed using the GROMACS webserver to determine the stability of mutants compared to the native protein. Co-expression, protein-protein interaction (PPI), gene ontology (GO), and pathway analyses were additionally performed for the CYP1B1 in-depth study. Results: All the retrieved data from the dbSNP database was subjected to functional, structural, and phylogenetic analysis. From the conducted analyses, a total of 19 high-risk variants (P52L, G61E, G90R, P118L, E173K, D291G, Y349D, G365W, G365R, R368H, R368C, D374N, N423Y, D430E, P442A, R444Q, F445L, R469W, and C470Y) were screened out that were considered to be deleterious to the CYP1B1 gene. The phylogenetic analysis revealed that the majority of the variants occurred in highly conserved regions. The MD simulation analysis exhibited that all mutants' average root mean square deviation (RMSD) values were higher compared to the wild-type protein, which could potentially cause CYP1B1 protein dysfunction, leading to the severity of the disease. Moreover, it has been discovered that CYP1A1, VCAN, HSD17B1, HSD17B2, and AKR1C3 are highly co-expressed and interact with CYP1B1. Besides, the CYP1B1 protein is primarily involved in the metabolism of xenobiotics, chemical carcinogenesis, the retinal metabolic process, and steroid hormone biosynthesis pathways, demonstrating its multifaceted and important roles. Discussion: This is the first comprehensive study that adds essential information to the ongoing efforts to understand the crucial role of genetic signatures in the development of PCG and will be useful for more targeted gene-disease association studies.

Phytochemicals and Biological Activities of Garcinia atroviridis: A Critical Review.

Garcinia atriviridis Griff ex T. Anders (G. atroviridis) is one of the well-known species of the genus Garicinia that is native to Thailand, Myanmar, Peninsular Malaysia, and India. G. atroviridis is a perennial medium-sized tree that has a wide range of values, from food to medicinal use. Different parts of G. atroviridis are a great source of bioactive substances that have a positive impact on health. The extracts or bioactive constituents from G. atroviridis have demonstrated various therapeutic functions, including antioxidant, antimicrobial, anticancer, anti-inflammatory, antihyperlipidemic, and anti-diabetic. In this paper, we provide a critical review of G. atroviridis and its bioactive constituents in the prevention and treatment of different diseases, which will provide new insight to explore its putative domains of research.

The complete chloroplast genome sequence of Durio oxleyanus (Malvaceae) and its phylogenetic position.

Durio oxleyanus (Griff) of Malvaceae is considered a natural heritage by the countries that produce it, including Peninsular Malaysia, Sumatra, and Borneo. Even though the species is regarded as a commercially valuable fruit, cultivation of this species is uncommon. The dwindling population of this species in the wild has put its survival in jeopardy. Conservation efforts are required for this species, which are limited. In this study, the complete chloroplast (cp) genome of D. oxleyanus was assembled and characterized as a genomic resource for conservation programs. The complete cp genome size was 164,831 bp in length, with a pair of inverted repeats of 23,782 bp each, separating the 96,446-bp large and the 20,823-bp small single copies. A total of 135 genes were predicted, which consisted of 90 protein-coding, 37 tRNA, and eight rRNA genes. The overall GC content was 35.8%. The phylogenetic analysis based on the maximum-likelihood and Bayesian inference methods revealed that D. oxleyanus is closely related to D. zibethinus. The genomic data obtained will be useful for future studies of Malvaceae's phylogenetics and evolution.

Marantodes pumilum: Systematic computational approach to identify their therapeutic potential and effectiveness.

ETHNOPHARMACOLOGICAL RELEVANCE: Marantodes pumilum (MP) herbs, locally known as Kacip Fatimah, are widely used traditionally to improve women's health. The herb is frequently used for gynecological issues such as menstrual problems, facilitating and quickening delivery, post-partum medication, treats flatulence and dysentery, and. MP extracts are thought to aid in the firming and toning of abdominal muscles, tighten breasts and vaginal muscles, and anti-dysmenorrhea. It also was used for the treatment of gonorrhea and hemorrhoids. As MP product has been produced commercially recently, more in-depth studies should be conducted. The presence of numerous active compounds in MP might provide a synergistic effect and potentially offer other health benefits than those already identified and known. AIM OF THE STUDY: This study aimed to use a computational target fishing approach to predict the possible therapeutic effect of Marantodes pumilum and evaluated their effectivity. MATERIALS AND METHODS: This study involves a computational approach to identify the potential targets by using target fishing. Several databases were used: PubChem database to obtain the chemical structure of interested compounds; Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) server and the SWISSADME web tool to identify and select the compounds having drug-likeness properties; PharmMapper was used to identify top ten target protein of the selected compounds and Online Mendelian Inheritance in Man (OMIM) was used to predict human genetic problems; the gene id of top-10 proteins was obtained from UniProtKB to be analyzed by using GeneMANIA server to check the genes' function and their co-expression; Gene Pathway established by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) of the selected targets were analyzed by using EnrichR server and confirmed by using DAVID (The Database for Annotation, Visualization and Integrated Discovery) version 6.8 and STRING database. All the interaction data was analyzed by Cytoscape version 3.7.2 software. The protein structure of most putative proteins was obtained from the RCSB protein data bank. Thedocking analysis was conducted using PyRx biological software v0.8 and illustrated by BIOVIA Discovery Studio Visualizer version 20.1.0. As a preliminary evaluation, a cell viability assay using Sulforhodamine B was conducted to evaluate the potential of the predicted therapeutic effect. RESULTS: It was found that four studied compounds are highly correlated with three proteins: EFGR, CDK2, and ESR1. These proteins are highly associated with cancer pathways, especially breast cancer and prostate cancer. Qualitatively, cell proliferation assay conducted shown that the extract has IC50 of 88.69 µg/ml against MCF-7 and 66.51 µg/ml against MDA-MB-231. CONCLUSIONS: Natural herbs are one of the most common forms of complementary and alternative medicine, and they play an important role in disease treatment. The results of this study show that in addition to being used traditionally to maintain women's health, the use of Marantodes pumilum indirectly has the potential to protect against the development of cancer cells, especially breast cancer. Therefore, further research is necessary to confirm the potential of this plant to be used in the development of anti-cancer drugs, especially for breast cancer.

In silico study of missense variants of FANCA, FANCC and FANCG genes reveals high risk deleterious alleles predisposing to Fanconi anemia pathogenesis.

Among the 22 Fanconi anemia (FA) reported genes, 90% of mutational spectra were found in three genes, namely FANCA (64%), FANCC (12%) and FANCG (8%). Therefore, this study aimed to identify the high-risk deleterious variants in three selected genes (FANCA, FANCC, and FANCG) through various computational approaches. The missense variant datasets retrieved from the UCSC genome browser were analyzed for their pathogenicity, stability, and phylogenetic conservancy. A total of 23 alterations, of which 16 in FANCA, 6 in FANCC and one variant in FANCG, were found to be highly deleterious. The native and mutant structures were generated, which demonstrated a profound impact on the respective proteins. Besides, their pathway analysis predicted many other pathways in addition to the Fanconi anemia pathway, homologous recombination, and mismatch repair pathways. Hence, this is the first comprehensive study that can be useful for understanding the genetic signatures in the development of FA.

Anti-breast cancer synthetic peptides derived from the Anabas testudineus skin mucus fractions.

Previous study has shown the antimicrobial activities of mucus protein extracted from Anabas testudineus. In this study, we are interested in characterizing the anticancer activity of the A. testudineus antimicrobial peptides (AMPs). The mucus was extracted, fractioned, and subjected to antibacterial activity testing to confirm the fish's AMPs production. The cytotoxic activity of each fraction was also identified. Fraction 2 (F2), which shows toxicity against MCF7 and MDA-MB-231 were sent for peptide sequencing to identify the bioactive peptide. The two peptides were then synthetically produced and subjected to cytotoxic assay to prove their efficacy against cancer cell lines. The IC50 for AtMP1 against MCF7 and MDA-MB-231 were 8.25 ± 0.14 µg/ml and 9.35 ± 0.25 µg/ml respectively, while for AtMP2 it is 5.89 ± 0.14 µg/ml and 6.97 ± 0.24 µg/ml respectively. AtMP1 and AtMP2 treatment for 48 h induced breast cancer cell cycle arrest and apoptosis by upregulating the p53, which lead to upregulate pro-apoptotic BAX gene and downregulate the anti-apoptotic BCL-2 gene, consequently, trigger the activation of the caspase-3. This interaction was supported by docking analysis (QuickDBD, HPEPDOCK, and ZDOCK) and immunoprecipitation. This study provided new prospects in the development of highly effective and selective cancer therapeutics based on antimicrobial peptides.

Mutated Shiitake extracts inhibit melanin-producing neural crest-derived cells in zebrafish embryo.

The ability of natural extracts to inhibit melanocyte activity is of great interest to researchers. This study evaluates and explores the ability of mutated Shiitake (A37) and wildtype Shiitake (WE) extract to inhibit this activity. Several properties such as total phenolic (TPC) and total flavonoid content (TFC), antioxidant activity, effect on cell and component profiling were conducted. While having no significant differences in total phenolic content, mutation resulted in A37 having a TFC content (1.04 ± 0.7 mg/100 ml) compared to WE (0.86 ± 0.9 mg/100 ml). Despite that, A37 extract has lower antioxidant activity (EC50, A37 = 549.6 ± 2.70 µg/ml) than WE (EC50 = 52.8 ± 1.19 µg/ml). Toxicity tests on zebrafish embryos show that both extracts, stop the embryogenesis process when the concentration used exceeds 900 µg/ml. Although both extracts showed pigmentation reduction in zebrafish embryos, A37 extract showed no effect on embryo heartbeat. Cell cycle studies revealed that WE significantly affect the cell cycle while A37 not. Further tests found that these extracts inhibit the phosphorylation of Glycogen synthase kinase 3 ß (pGSK3ß) in HS27 cell line, which may explain the activation of apoptosis in melanin-producing cells. It was found that from 19 known compounds, 14 compounds were present in both WE and A37 extracts. Interestingly, the presence of decitabine in A37 extract makes it very potential for use in the medical application such as treatment of melanoma, skin therapy and even cancer.

Comprehensive computational target fishing approach to identify Xanthorrhizol putative targets.

Xanthorrhizol (XNT), is a bioactive compound found in Curcuma xanthorrhiza Roxb. This study aimed to determine the potential targets of the XNT via computational target fishing method. This compound obeyed Lipinski's and Veber's rules where it has a molecular weight (MW) of 218.37 gmol-1, TPSA of 20.23, rotatable bonds (RBN) of 4, hydrogen acceptor and donor ability is 1 respectively. Besides, it also has half-life (HL) values 3.5 h, drug-likeness (DL) value of 0.07, oral bioavailability (OB) of 32.10, and blood-brain barrier permeability (BBB) value of 1.64 indicating its potential as therapeutic drug. Further, 20 potential targets were screened out through PharmMapper and DRAR-CPI servers. Co-expression results derived from GeneMANIA revealed that these targets made connection with a total of 40 genes and have 744 different links. Four genes which were RXRA, RBP4, HSD11B1 and AKR1C1 showed remarkable co-expression and predominantly involved in steroid metabolic process. Furthermore, among these 20 genes, 13 highly expressed genes associated with xenobiotics by cytochrome P450, chemical carcinogenesis and steroid metabolic pathways were identified through gene ontology (GO) and KEGG pathway analysis. In conclusion, XNT is targeting multiple proteins and pathways which may be exploited to shape a network that exerts systematic pharmacological effects.

Cytotoxicity and Toxicity Evaluation of Xanthone Crude Extract on Hypoxic Human Hepatocellular Carcinoma and Zebrafish (Danio rerio) Embryos.

Xanthone is an organic compound mostly found in mangosteen pericarp and widely known for its anti-proliferating effect on cancer cells. In this study, we evaluated the effects of xanthone crude extract (XCE) and α-mangostin (α-MG) on normoxic and hypoxic human hepatocellular carcinoma (HepG2) cells and their toxicity towards zebrafish embryos. XCE was isolated using a mixture of acetone and water (80:20) and verified via high performance liquid chromatography (HPLC). Both XCE and α-MG showed higher anti-proliferation effects on normoxic HepG2 cells compared to the control drug, 5-fluorouracil (IC50 = 50.23 ± 1.38, 8.39 ± 0.14, and 143.75 ± 15.31 µg/mL, respectively). In hypoxic conditions, HepG2 cells were two times less sensitive towards XCE compared to normoxic HepG2 cells (IC50 = 109.38 ± 1.80 µg/mL) and three times less sensitive when treated with >500 µg/mL 5-fluorouracil (5-FU). A similar trend was seen with the α-MG treatment on hypoxic HepG2 cells (IC50 = 10.11 ± 0.05 µg/mL) compared to normoxic HepG2 cells. However, at a concentration of 12.5 µg/mL, the α-MG treatment caused tail-bend deformities in surviving zebrafish embryos, while no malformation was observed when embryos were exposed to XCE and 5-FU treatments. Our study suggests that both XCE and α-MG are capable of inhibiting HepG2 cell proliferation during normoxic and hypoxic conditions, more effectively than 5-FU. However, XCE is the preferred option as no malformation was observed in surviving zebrafish embryos and it is more cost efficient than α-MG.

The Glaciozyma antarctica genome reveals an array of systems that provide sustained responses towards temperature variations in a persistently cold habitat.

Extremely low temperatures present various challenges to life that include ice formation and effects on metabolic capacity. Psyhcrophilic microorganisms typically have an array of mechanisms to enable survival in cold temperatures. In this study, we sequenced and analysed the genome of a psychrophilic yeast isolated in the Antarctic region, Glaciozyma antarctica. The genome annotation identified 7857 protein coding sequences. From the genome sequence analysis we were able to identify genes that encoded for proteins known to be associated with cold survival, in addition to annotating genes that are unique to G. antarctica. For genes that are known to be involved in cold adaptation such as anti-freeze proteins (AFPs), our gene expression analysis revealed that they were differentially transcribed over time and in response to different temperatures. This indicated the presence of an array of adaptation systems that can respond to a changing but persistent cold environment. We were also able to validate the activity of all the AFPs annotated where the recombinant AFPs demonstrated anti-freeze capacity. This work is an important foundation for further collective exploration into psychrophilic microbiology where among other potential, the genes unique to this species may represent a pool of novel mechanisms for cold survival.

Flo11p adhesin required for meiotic differentiation in Saccharomyces cerevisiae minicolonies grown on plastic surfaces.

Saccharomyces cerevisiae grown on plastic surfaces formed organized structures, termed minicolonies, that consisted of a core of round (yeast-like) cells surrounded by chains of filamentous cells (pseudohyphae). Minicolonies had a much higher affinity for plastic than unstructured yeast communities growing on the same surface. Pseudohyphae at the surface of these colonies developed further into chains of asci. These structures suggest that pseudohyphal differentiation and sporulation are sequential processes in minicolonies. Consistent with this idea, minicolonies grown under conditions that stimulated pseudohyphal differentiation contained higher frequencies of asci. Furthermore, a flo11Δ mutant, which fails to form pseudohyphae, yielded normal sporulation in cultures, but was defective for minicolony sporulation. When minicolonies were dispersed in water and cells were then allowed to settle on the plastic surface, these cells sporulated very efficiently. Taken together, our results suggest that sporulation in minicolonies is stimulated by pseudohyphal differentiation because these pseudohyphae are dispersed from the core of the colony.

The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.

Multicellular organisms utilize cell-to-cell signals to build patterns of cell types within embryos, but the ability of fungi to form organized communities has been largely unexplored. Here we report that colonies of the yeast Saccharomyces cerevisiae formed sharply divided layers of sporulating and nonsporulating cells. Sporulation initiated in the colony's interior, and this region expanded upward as the colony matured. Two key activators of sporulation, IME1 and IME2, were initially transcribed in overlapping regions of the colony, and this overlap corresponded to the initial sporulation region. The development of colony sporulation patterns depended on cell-to-cell signals, as demonstrated by chimeric colonies, which contain a mixture of two strains. One such signal is alkaline pH, mediated through the Rim101p/PacC pathway. Meiotic-arrest mutants that increased alkali production stimulated expression of an early meiotic gene in neighboring cells, whereas a mutant that decreased alkali production (cit1Delta) decreased this expression. Addition of alkali to colonies accelerated the expansion of the interior region of sporulation, whereas inactivation of the Rim101p pathway inhibited this expansion. Thus, the Rim101 pathway mediates colony patterning by responding to cell-to-cell pH signals. Cell-to-cell signals coupled with nutrient gradients may allow efficient spore formation and spore dispersal in natural environments.

Improvement in survival and muscle function in an mdx/utrn(-/-) double mutant mouse using a human retinal dystrophin transgene.

Duchenne muscular dystrophy is a progressive muscle disease characterized by increasing muscle weakness and death by the third decade. mdx mice exhibit the underlying muscle disease but appear physically normal with ordinary lifespans, possibly due to compensatory expression of utrophin. In contrast, double mutant mice (mdx/utrn(-/-)), deficient for both dystrophin and utrophin die by approximately 3 months and suffer from severe muscle weakness, growth retardation, and severe spinal curvature. The capacity of human retinal dystrophin (Dp260) to compensate for the missing 427 kDa muscle dystrophin was tested in mdx/utrn(-/-) mice. Functional outcomes were assessed by histology, EMG, MRI, mobility, weight and longevity. MCK-driven transgenic expression of Dp260 in mdx/utrn(-/-) mice converts their disease course from a severe, lethal muscular dystrophy to a viable, mild myopathic phenotype. This finding is relevant to the design of exon-skipping therapeutic strategies since Dp260 lacks dystrophin exons 1-29.

Extracellular bone acidic glycoprotein-75 defines condensed mesenchyme regions to be mineralized and localizes with bone sialoprotein during intramembranous bone formation.

Bone acidic glycoprotein-75 is expressed very early during in vivo models of intramembranous bone formation, highly enriched in condensing osteogenic mesenchyme after marrow ablation and the osteoprogenitor layer of tibial periosteum. Bone sialoprotein accumulates within bone acidic glycoprotein-75-enriched matrix areas at a later stage in both models. Decalcification of initial sites of mineralization consistently revealed focal immunostaining for bone acidic glycoprotein-75 underneath these sites suggesting that mineralization occurs within bone acidic glycoprotein-75-enriched matrix areas. Ultrastructural immunolocalization of bone acidic glycoprotein-75 does not support a direct association with banded collagen fibrils, but rather suggests it is a component of a separate, amorphous scaffold occupying interfibrillar spaces. Double immunogold labeling demonstrated that a sizeable proportion of bone sialoprotein particles were located within a 50-nm radius of bone acidic glycoprotein-75. These results define bone acidic glycoprotein-75 as the earliest bone-restricted, extracellular marker of osteogenic mesenchyme. Based on this early bone-restricted expression pattern and a previously documented propensity of bone acidic glycoprotein-75 to form supramolecular complexes through self-association, bone acidic glycoprotein-75 may serve a key structural role in setting boundary limits of condensing osteogenic mesenchyme.

Bone acidic glycoprotein-75 delineates the extracellular sites of future bone sialoprotein accumulation and apatite nucleation in osteoblastic cultures.

Addition of an organophosphate source to UMR osteoblastic cultures activates a mineralization program in which BSP localizes to extracellular matrix sites where hydroxyapatite crystals are subsequently nucleated. This study identifies for the first time novel extracellular spherical structures, termed biomineralization foci (BMF), containing bone acidic glycoprotein-75 (BAG-75), bone sialoprotein (BSP), and alkaline phosphatase that are the exclusive sites of initial nucleation of hydroxyapatite crystals in the UMR model. Importantly, in the absence of added phosphate, UMR cultures after reaching confluency contain two size populations of morphologically identifiable BMF precursors enriched in BAG-75 (15-25 and 150-250 microm in diameter). The shape and size of the smaller population are similar to structures assembled in vitro through self-association of purified BAG-75 protein. After organophosphate addition, BSP accumulates within these BAG-75-containing BMF precursors, with hydroxyapatite crystal nucleation occurring subsequently. In summary, BAG-75 is the earliest detectable biomarker that accurately predicts the extracellular sites of de novo biomineralization in UMR cultures. We hypothesize that BAG-75 may perform a key structural role in the assembly of BMF precursors and the recruitment of other proteins such as alkaline phosphatase and BSP. Furthermore, we propose a hypothetical mechanism in which BAG-75 and BSP function actively in nucleation of apatite within BMF.