A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation

Objective: Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels. Design: hFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays. Results: LMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in Vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day (p < 0.001) of culturing, decreasing thereafter. Conclusions: Our results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. in this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation. (C) 2009 Elsevier Ltd. All rights reserved.

Effect of growth hormone on in vitro osteogenesis and gene expression of human osteoblastic cells is donor-age-dependent

it has been demonstrated that the effect of GH on bone tissue is reduced with aging. In this study we tested the hypothesis that the action of GH on osteoblastic cells is donor-age-dependent by investigating the effect of GH on the development of osteoblastic phenotype in cultures of cells from adolescents (13-16 years old), young adults (18-35 years old), and adults (36-49 years old). Osteoblastic cells derived from human alveolar bone were cultured with or without GH for periods of up to 21 days, and parameters of in vitro osteogenesis and gene expression of osteoblastic markers were evaluated. GH increased culture growth, collagen content and alkaline phosphatase (ALP) activity in cultures from adolescents and young adults, whereas non-significant effect was observed in cultures from adults. While GH significantly increased the bone-like formation in cultures from adolescents, a slightly effect was observed in cultures from young adults and no alteration was detected in cultures from adults. Results from real-time PCR demonstrated that GH upregulated ALP, osteocalcin, type I collagen, and Cbfa1 mRNA levels in cultures from adolescents. In addition, cultures from young adults showed higher ALP mRNA expression and the expression of all evaluated genes was not affected by GH in cultures from adults. These results indicate that the GH effect on both in vitro osteogenesis and gene expression of osteoblastic markers is donor-age-dependent, being more pronounced on cultures from adolescents.

Assessment of DGAT1 and LEP gene polymorphisms in three Nelore (Bos indicus) lines selected for growth and their relationship with growth and carcass traits

The aim of this study was to analyze LEP and DGAT1 gene polymorphisms in 3 Nelore lines selected for growth and to evaluate their effects on growth and carcass traits. Traits analyzed were birth, weaning, and yearling weight, rump height, LM area, backfat thickness, and rump fat thickness obtained by ultrasound. Two SNP in the LEP gene [LEP 1620(A/G) and LEP 305(T/C)] and the K232A mutation in the DGAT1 gene were analyzed. The sample consisted of 357 Nelore heifers from 2 lines selected for yearling weight and a control line, established in 1980, at the Estacao Experimental de Zootecnia de Sertaozinho (Sertaozinho, Brazil). Three genotypes were obtained for each marker. Differences in allele frequencies among the 3 lines were only observed for the DGAT1 K232A polymorphism, with the frequency of the A allele being greater in the control line than in the selected lines. The DGAT1 K232A mutation was associated only with rump height, whereas LEP 1620(A/G) was associated with weaning weight and LEP 305(T/C) with birth weight and backfat thickness. However, more studies, with larger data sets, are necessary before these makers can be used for marker-assisted selection.

Life history, morphological variability and growth rates of the life phases of Gracilaria tenuistipitata (Rhodophyta: Gracilariales) in vitro

Gracilaria tenuistipitata, a species of commercial interest, is becoming a model organism for studies on red algal physiology and molecular biology as it can be grown easily in vitro under a broad range of conditions. Most of the experiments carried out around the world have been based on a tetrasporophytic clone isolated in our laboratory from a specimen collected in China. Here we describe the life history of this species, give anatomic details of the reproductive structures, illustrate the morphological variability of tetraspore progeny and compare the growth rate of gametophytic and sporophytic thalli. Tetrasporophytic branches showed higher growth rates than gametophytic branches.

Nitrosyl ethylenediaminetetraacetate ruthenium(II) complex promotes cellular growth and could be used as nitric oxide donor in plants

Over recent years nitric oxide (NO) not only has appeared as an important endogenous signaling molecule in plants and as a mediator in many developmental and physiological processes, but has also received recognition as a plant hormone. The impressive recent achievements in elucidating the role of NO in plants have come about by the application of NO donors. The aim herein was to study the effects of the different NO donors, sodium nitroprusside (SNP) and the nitrosyl ethylenediaminetetraacetate ruthenium(II) ([Ru(NO)(Hedta)]) complex on cellular growth in embryogenic suspension cultures of Araucaria angustifolia. Appraisal of our data revealed that [Ru(NO)(Hedta)] stimulated about 60% of cellular growth in embryogenic suspension cultures of A. angustifolia, with results similar to those observed with the SNP donor. Nevertheless, application of the NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) inhibited this cellular growth in both. Cellular growth was correlated with an increase in endogenous NO levels after 21 days of culture, especially in treatments with NO donors. Our results demonstrated that the [Ru(NO)Hedta] complex could possibly be used as a NO donor in plants. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Potential Role of Growth Hormone in Impairment of Insulin Signaling in Skeletal Muscle, Adipose Tissue, and Liver of Rats Chronically Treated with Arginine

We have shown that rats chronically treated with Arginine (Arg), although normoglycemic, exhibit hyperinsulinemia and decreased blood glucose disappearance rate after an insulin challenge. Attempting to investigate the processes underlying these alterations, male Wistar rats were treated with Arg (35 mg/d), in drinking water, for 4 wk. Rats were then acutely stimulated with insulin, and the soleus and extensorum digitalis longus muscles, white adipose tissue (WAT), and liver were excised for total and/or phosphorylated insulin receptor (IR), IR substrate 1/2, Akt, Janus kinase 2, signal transducer and activator of transcription (STAT) 1/3/5, and p85 alpha/55 alpha determination. Muscles and WAT were also used for plasma membrane (PM) and microsome evaluation of glucose transporter (GLUT) 4 content. Pituitary GH mRNA, GH, and liver IGF-I mRNA expression were estimated. It was shown that Arg treatment: 1) did not affect phosphotyrosine-IR, whereas it decreased phosphotyrosine-IR substrate 1/2 and phosphoserine-Akt content in all tissues studied, indicating that insulin signaling is impaired at post-receptor level; 2) decreased PM GLUT4 content in both muscles and WAT; 3) increased the pituitary GH mRNA, GH, and liver IGF-I mRNA expression, the levels of phosphotyrosine-STAT5 in both muscles, phosphotyrosine-Janus kinase 2 in extensorum digitalis longus, phosphotyrosine-STAT3 in liver, and WAT as well as total p85 alpha in soleus, indicating that GH signaling is enhanced in these tissues; and 4) increased p55 alpha total content in muscles, WAT, and liver. The present findings provide the molecular mechanisms by which insulin resistance and, by extension, reduced GLUT4 content in PM of muscles and WAT take place after chronic administration of Arg, and further suggest a putative role for GH in its genesis, considering its diabetogenic effect. (Endocrinology 150: 2080-2086, 2009)

Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour

Aims: Ameloblastoma is an odontogenic neoplasm with local invasiveness and recurrence. We have previously suggested that growth factors and matrix metalloproteinases (MMPs) influence ameloblastoma invasiveness(1). The aim was to study expression of MMPs, tissue inhibitor of metalloproteinases (TIMPs) and growth factors in ameloblastoma. Methods and results: Thirteen cases of solid/multicystic ameloblastoma were examined. As a control, calcifying cystic odontogenic tumour (CCOT), a non-invasive odontogenic neoplasm with ameloblastomatous epithelium was also studied. Immunohistochemistry detected MMPs, TIMPs and growth factors in ameloblastoma and CCOT. The labelling index (LI) of MMP-9 and TIMP-2 was significantly higher in ameloblastoma compared with CCOT. The LI of epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) was also increased in ameloblastoma. This neoplasm showed greater expression of MMPs, TIMPs and growth factors compared with CCOT. We then analysed these molecules in ameloblastoma cells and stroma. Ameloblastoma cells exhibited increased LI of MMP-1, -2 and EGFR. We found a positive correlation between EGF and TIMP-1, and between TGF-alpha and TIMP-2. It is known that signals generated by growth factors are transduced by the ERK pathway. Ameloblastoma stroma exhibited the phosphorylated (activated) form of ERK. Conclusions: These results suggest an interplay involving growth factors MMPs and TIMPs that may contribute to ameloblastoma behaviour. Signals generated by this molecular network would be transduced by ERK 1/2 pathway.

Effects of let-7 microRNA on Cell Growth and Differentiation of Papillary Thyroid Cancer

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy and RET/PTC rearrangements represent key genetic events frequently associated to this cancer, enhancing proliferation and dedifferentiation by activation of the RET/PTC-RAS-BRAF-mitogen-activated protein kinase (MAPK) pathway. Recently, let-7 microRNA was found to reduce RAS levels in lung cancer, acting as a tumor suppressor gene. Here, we report that RET/PTC3 oncogenic activation in PCCL3 rat thyroid cells markedly reduces let-7f expression. Moreover, stable transfection of let-7 microRNA in TPC-1 cells, which harbor RET/PTC1 rearrangement, inhibits MAPK activation. As a result, let-7f was capable of reducing TPC-1 cell growth, and this might be explained, at least in part, by decreased messenger RNA (mRNA) expression of cell cycle stimulators such as MYC and CCND1 (cyclin D1) and increased P21 cell cycle inhibitor mRNA. In addition, let-7 enhanced transcriptional expression of molecular markers of thyroid differentiation such as TITF1 and TG. Thus, reduced expression of let-7f might be an essential molecular event in RET/PTC malignant transformation. Moreover, let-7f effects on thyroid growth and differentiation might attenuate neoplastic process of RET/PTC papillary thyroid oncogenesis through impairment of MAPK signaling pathway activation. This is the first functional demonstration of an association of let-7 with thyroid cancer cell growth and differentiation.

Resistance to ultraviolet-induced apoptosis in DNA repair deficient growth arrested human fibroblasts is not related to recovery from RNA transcription blockage

The impact of ultraviolet (UV-C) photoproducts on apoptosis induction was investigated in growth arrested (confluent) and proliferating human primary fibroblasts. Confluent fibroblasts were more resistant to UV-C-induced apoptosis than proliferating cells, and this was observed for normal human cells and for cells from patients with Cockayne and trichothiodystrophy syndromes, deficient in transcription coupled repair. This resistance was sustained for at least seven days and was not due to DNA repair efficiency, as the removal of CPDs in the genome was similar under both growth conditions. There was no correlation between reduced apoptosis and RNA synthesis recovery. Following UV-C treatment, proliferating and confluent fibroblasts showed a similar level of RNA synthesis inhibition and recovery from transcription blockage. These results support the hypothesis that the decrease of DNA replication, in growth arrested cells, protects cell from UV-C-induced apoptosis, even in the presence of DNA lesions. (C) 2007 Elsevier B.V. All rights reserved.

Phylogeny of snake trypanosomes inferred by SSU rDNA sequences, their possible transmission by phlebotomines, and taxonomic appraisal by molecular, cross-infection and morphological analysis

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 24% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannaniyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies dagger. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor kappa B ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers` expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth. Copyright (C) 2007 John Wiley & Sons, Ltd.

Infrared analysis of ion beam irradiated polymers

Irradiation with heavy ions can produce several modifications in the chain structure of polymers. These modifications can be related to scissioning and cross-linking of chemical bonds. which depend on the ion fluence and the density of energy deposited in the material. Stacked thin film Makrofol-KG (R) samples were irradiated with 350 MeV Au(26+) ions and FTIR absorption spectroscopy was used to determine the bond changes in the samples. Data on the absorption bands as a function of the fluence indicated a higher probability for simple-bonds scissioning than for double-bonds scissioning and no dependence on the number of double bonds breaking with ion fluence. Since sample irradiation was done in a non-track-overlapping regime, a novel process for double bonds formation is suggested: the excitation of a site in the material by only one incident ion followed by a double bond formation during the de-excitation process. (C) 2009 Elsevier B.V. All rights reserved.

Nonlinear spectrum effect on the coherent control of molecular systems

This work demonstrates that the detuning of the fs-laser spectrum from the two-photon absorption band of organic materials can be used to reach further control of the two-photon absorption by pulse spectral phase manipulation. We investigate the coherent control of the two-photon absorption in imidazole-thiophene core compounds presenting distinct two-photon absorption spectra. The coherent control, performed using pulse phase shaping and genetic algorithm, exhibited different growth rates for each sample. Such distinct trends were explained by calculating the two-photon absorption probability considering the intrapulse interference mechanism, taking into account the two-photon absorption spectrum of the samples. Our results indicate that tuning the relative position between the nonlinear absorption and the pulse spectrum can be used as a novel strategy to optimize the two-photon absorption in broadband molecular systems. (C) 2011 Elsevier B.V. All rights reserved.

Directed self-assembly of quantum structures by nanomechanical stamping using probe tips

We demonstrate that nanomechanically stamped substrates can be used as templates to pattern and direct the self-assembly of epitaxial quantum structures such as quantum dots. Diamond probe tips are used to indent or stamp the surface of GaAs( 100) to create nanoscale volumes of dislocation-mediated deformation, which alter the growth surface strain. These strained sites act to bias nucleation, hence allowing for selective growth of InAs quantum dots. Patterns of quantum dots are observed to form above the underlying nanostamped template. The strain state of the patterned structures is characterized by micro-Raman spectroscopy. The potential of using nanoprobe tips as a quantum dot nanofabrication technology are discussed.

Crystal Engineering of an Anti-HIV Drug Based on the Recognition of Assembling Molecular Frameworks

A rational strategy was employed for design of an orthorhombic structure of lamivudine with maleic acid. On the basis of the lamivudine saccharinate structure reported in the literature, maleic acid was chosen to synthesize a salt with the anti-HIV drug because of the structural similarities between the salt formers. Maleic acid has an acid-ionization constant of the anti first proton and an arrangement of their hydrogen bonding functionalities similar to those of saccharin. Likewise, there is a saccharin-like conformational rigidity in maleic acid because of the hydrogen-bonded ring formation and the Z-configuration around the C=C double bond. As was conceivably predicted, lamivudine maleate assembles into a structure whose intermolecular architecture is related to that of saccharinate salt of the drug. Therefore, a molecular framework responsible for crystal assembly into a lamivudine saccharinate-like structure could be recognized in the salt formers. Furthermore, structural correlations and structure-solubility relationships were established for lamivudine maleate and saccharinate. Although there is a same molecular framework in maleic acid and saccharin, these salt formers are Structurally different in some aspects. When compared to saccharin, neither out-of-plane SO(2) oxygens nor a benzene group occur in maleic acid. Both features could be related to higher solubility of lamivudine maleate. Here, we also anticipate that multicomponent molecular crystals of lamivudine with other salt formers possessing the molecular framework responsible for crystal assembly can be engineered successfully.