Specific and rapid detection of proteins in biological fluids poses a challenging problem. In biological fluids, many proteins are present at low concentrations, requiring high affinity and ...specificity of the beacon‐protein interaction. We report the design of a peptide‐PNA hybrid beacon that exploits the dimeric nature of a target protein, S100B, a biomarker for brain trauma, to enhance binding affinity and specificity. The complementary base‐pairing of the PNA bases brings the two arms of the beacon, one carrying an Alexa tag and the other carrying a Dabcyl moiety, into proximity, thus quenching Alexa fluorescence. Each of the arms carries a sequence that binds to one of the subunits. Binding to the target separates the quencher from the probe lifting the quenching of fluorescence. Enhanced affinity and specificity resulting from simultaneously binding to two sites allowed specific detection of S100B at low‐nanomolar concentrations in the presence of serum. The design can be easily adapted for the detection of proteins containing multiple binding sites and could prove useful for rapid and sensitive biomarker detection.
Needle in a haystack: A fluorescence‐based peptide‐PNA hybrid beacon targeting the S100B protein, a biomarker for brain trauma, has been developed. The molecular beacon is capable of detecting nanomolar concentrations of S100B protein rapidly, even in the presence of serum. The design should be useful for developing molecular beacons for other protein biomarkers.
Background The authenticity of various prototype human placental extracts with biological activity, such as that inducing vitiligo repigmentation, is under serious criticism, mainly due to a lack of ...demonstration at the cellular level. Considering the present worldwide scenario with regard to the occurrence and treatment of vitiligo, a thorough scientific exploration of such extracts should be undertaken.
Method One such prototype placental preparation was prepared, and was evaluated with regard to its melanogenic action in C57BL/6J mice in vivo and its mitogenic and melanogenic activity on B16F10 mouse melanoma cells and normal human melanocytes in vitro. The extract was applied topically to mice with age‐induced prolonged telogenic phase of hair growth (grey body coat hair). Standard 3H‐thymidine incorporation and spectrophotometric methods were followed to illustrate mitogenic and melanogenic effects at the cellular level.
Results The resurgence of blue skin, followed by shiny black hair, at the regions of application of the extract demonstrated the reversal of the age‐induced prolonged telogenic phase of hair growth to the anagenic phase after topical application of the extract on C57BL/6J mice. Further support was obtained from histology where, at the extract‐treated sites, the development of new melanogenic centers and hair follicles was observed. During in vitro studies, the vehicle‐free extract constituents stimulated both mitogenesis and melanogenesis of B16F10 mouse melanoma cells in a concentration‐dependent manner. The cell morphology and extent of melanogenesis also showed significant changes. In addition, two known melanocyte activity‐modulating peptides, endothelin‐1 (ET‐1) and adrenocorticotropic hormone (ACTH), were determined in the extract, chiefly in the total lipid fraction, indicating their effective cutaneous permeation.
Conclusions The extract was found to be a potent mitogen in the in vitro condition and a potent melanogen in both the in vitro and in vivo situations. This strongly suggests its therapeutic potential for the repigmentation of vitiligo patches.
Summary
Recent advances in the identification and characterisation of stem cell populations has led to substantial interest in understanding the precise triggers that would operate to induce ...activation of quiescent stem cells. Melanocyte stem cells (MSCs) reside in the bulge region of the hair follicles and are characterised by reduced expression of the microphthalmia‐associated transcription factor (Mitf) and its target genes implicated in differentiation. Vitiligo is characterised by progressive destruction of differentiated melanocytes. However, therapies using UV irradiation therapy can induce a degree of repigmentation, suggesting that MSCs may be activated. As Mitf is implicated in control of proliferation, we have explored the possibility that inducing Mitf expression via lipid‐mediated activation of the p38 stress‐signalling pathway may represent a re‐pigmentation strategy. Here we have isolated from placental extract a C18:0 sphingolipid able to induce Mitf and tyrosinase expression via activation of the p38 stress‐signalling pathway. Strikingly, in age‐onset gray‐haired C57BL/6J mice that exhibit decaying Mitf expression, topical application of placental sphingolipid leads to increased Mitf in follicular melanocytes and fresh dense black hair growth. The results raise the possibility that lipid‐mediated activation of the p38 pathway may represent a novel approach to an effective vitiligo therapy.
Summary
Melanogenesis is one of the characteristic functional activities of melanocyte/melanoma and is regulated via mitogen‐activated protein kinase (MAPK) and Akt/protein kinase B (PKB) pathways. ...Placental total lipid fraction (PTLF), prepared from a hydroalcoholic extract of fresh term human placenta contains sphingolipids and was recently shown to stimulate melanogenesis via up‐regulation of the key enzyme tyrosinase in B16F10 mouse melanoma cells. How such lipids mediate their effects on pigmentation and tyrosinase expression is a particularly important aspect of melanogenesis. To study the signaling that leads to tyrosinase expression, we have investigated the roles of the MAPK and Akt/PKB pathways in B16F10 melanoma cells in melanogenesis in response to PTLF. Treatment of cells with PTLF led to the time dependent phosphorylation of p38 MAPK. SB203580, a p38 MAPK inhibitor, completely blocked the PTLF‐induced melanogenesis by inhibiting promoter activity and subsequent expression of tyrosinase. Phosphatidylinositol 3‐kinase (PI3K) inhibitor, LY294002 a blocker of the Akt signaling pathway, or an inhibitor of MEK (MAPK/ERK Kinase), PD98059 when included along with PTLF was found to potentiate PTLF‐induced phosphorylation of p38 MAPK together with tyrosinase expression and melanogenesis. The results suggest that the activation of p38 MAPK plays a crucial role in PTLF‐induced B16F10 melanogenesis by up‐regulating tyrosinase expression.
The sphingolipids, a class of complex bioactive lipids, are involved in diverse cellular functions such as proliferation, differentiation, and apoptosis as well as growth inhibition. Recently ...sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and C2-ceramide (C2-Cer), sphingolipid containing acetic acid are emerging as melanogenic regulators. A bioactive sphingolipid (PSL) was isolated from hydroalcoholic extract of fresh term human placenta and it induced melanogenesis in an in vitro culture of mouse melanoma B16F10 cells. Tyrosinase, the rate-limiting enzyme for melanogenesis, is required to be upregulated for the increased melanin production. The expression of tyrosinase, both at protein as well as mRNA level, was higher in the PSL treated B16F10 cells as evidenced by Western blot and RT-PCR analysis. Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, inhibited PSL-induced tyrosinase activity and its protein expression showing decrease in melanogenesis, correspondingly. The activity of GFP coupled tyrosinase promoter was upregulated in transfected B16F10 cells after treating with PSL as determined by fluorescence microscopy, fluorometric analysis, and Western blot. These results, thus, suggested that PSL upregulated tyrosinase gene expression at transcription level through promoter activation to show increased melanogenesis. Therefore, PSL as an inducer of melanogenesis might account for the recovery of pigment in depigmentation disorder.
Summary
Lipids, particularly sphingolipids, are emerging as novel regulators of cellular activity. A placental total lipid fraction (PTLF), the total lipid prepared from an hydroalcoholic extract of ...fresh term human placenta, was previously shown to have a pigment‐inducing activity in an animal model. The PTLF contains sphingolipids which stimulate DNA synthesis and melanin formation with marked morphological changes in B16F10 melanoma cells. In order to identify the mechanism underlying the increased melanin synthesis, B16F10 cells were treated with PTLF to assess the catalytic activities of tyrosinase (i.e. tyrosine hydroxylase and DOPA oxidase), the key regulatory enzyme of melanin synthesis. Tyrosine hydroxylase (estimated by the release of 3H2O) as well as DOPA oxidase (measured spectrophotometrically and also in non‐denaturing gels), was stimulated significantly by PTLF. Western blot analysis demonstrated an increase in the expression of tyrosinase, tyrosinase related proteins 1 and 2 (TRP1 and TRP2) at the protein level and RT‐PCR analysis revealed stimulated transcription of tyrosinase, TRP1 and TRP2 mRNAs in PTLF‐treated B16F10 cells. Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, inhibited PTLF‐induction of tyrosinase activity with a corresponding decrease in melanogenesis. In all cases, the response to PTLF was similar to that induced by α‐melanocyte stimulating hormone, a well‐known stimulator of melanogenesis. Thus, these results provide the basis of action of PTLF stimulated melanogenesis in B16F10 cells showing that this placental extract is a strong inducer of pigmentation at the transcriptional and translational levels.
A hydroalcoholic extract of fresh term human placenta was found to be mitogenic as well as melanogenic on B16F10 mouse melanoma in an in vitro culture. The extract, a reservoir of a large number of ...bioactive molecules, was resolved to get the lipid fraction. Its activity was evaluated on B16F10 mouse melanoma by assessing the change in cellular morphology, growth and melanin induction. The lipid fraction, placental total lipid fraction (PTLF) tested in the study employed doses of 0 01 to 200 microg/ml; optimum growth and melanization accompanied by morphological changes were recorded at 10 and 100 microg/ml respectively. At intermediate doses growth and melanization were found to show a pattern of change over between growth and melanization and finally reached at an inverse relation at the respective optimal dose of response. Compared with defined sphingolipids, C(2) ceramide and sphingosine-1-phosphate, the results were mostly corroborative. The duality of biological response of sphingolipids as reported in numerous studies was comparable for the PTLF suggesting that its active component is a sphingolipid and showing its use for pigment recovery in vitiligo.
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