α-Pinene, a Main Component of Pinus Essential Oils, Enhances the Expression of Insulin-Sensitive Glucose Transporter Type 4 in Murine Skeletal Muscle Cells.

Glucose transporter-4 (GLUT4) represents the major glucose transporter isoform responsible for glucose uptake into insulin-sensitive cells, primarily in skeletal muscle and adipose tissues. In insulin-resistant conditions, such as type 2 diabetes mellitus, GLUT4 expression and/or translocation to the cell plasma membrane is reduced, compromising cell energy metabolism. Therefore, the use of synthetic or naturally occurring molecules able to stimulate GLUT4 expression represents a good tool for alternative treatments of insulin resistance. The present study aimed to investigate the effects of essential oils (EOs) derived from Pinus spp. (P. nigra and P. radiata) and of their main terpenoid constituents (α- and ß-pinene) on the expression/translocation of GLUT4 in myoblast C2C12 murine cells. For this purpose, the chemical profiles of the EOs were first analyzed through gas chromatography-mass spectrometry (GC-MS). Cell viability was assessed by MTT assay, and GLUT4 expression/translocation was evaluated through RT-qPCR and flow cytometry analyses. The results showed that only the P. nigra essential oil (PnEO) and α-pinene can increase the transcription of the Glut4/Scl2a4 gene, resulting in a subsequent increase in the amount of GLUT4 produced and its plasma membrane localization. Moreover, the PnEO or α-pinene can induce Glut4 expression both during myogenesis and in myotubes. In summary, the PnEO and α-pinene emulate insulin's effect on the GLUT4 transporter expression and its translocation to the muscle cell surface.

Cytotoxicity of Isoxazole Curcumin Analogs on Chronic Myeloid Leukemia-Derived K562 Cell Lines Sensitive and Resistant to Imatinib.

Despite curcumin (CUR) inhibiting cell proliferation in vitro by activating apoptotic cell death, its use in pharmacological therapy is hampered by poor solubility, low stability in biological fluids, and rapid removal from the body. Therefore, CUR-derivatives with better biological and chemical-physical characteristics are needed. The bis-ketone moiety of CUR strongly influences its stability in slightly alkaline solutions such as plasma. Here, we considered its replacement with isoxazole, beta-enamine, or oxime groups to obtain more stable derivatives. The evaluation of the chemical-physical characteristics showed that only of the isoxazole derivatives 2 and 22 had better potential than CUR in terms of bioavailability. The UV-visible spectrum analysis showed that derivatives 2 and 22 had better stability than CUR in solutions mimicking the biological fluids. When tested on a panel of cell lines, derivatives 2 and 22 had marked cytotoxicity (IC50 = 0.5 µM) compared with CUR only (IC50 = 17 µM) in the chronic myeloid leukemia (CML)-derived K562 cell line. The derivative 22 was the more selective for CML cells. When administered at the average concentration found for CUR in the blood of patients, derivatives 2 and 22 had potent effects on cell cycle progression and apoptosis initiation, while CUR was ineffective. The apoptotic effect of derivatives 2 and 22 was associated with low necrosis. In addition, derivative 22 was able to reverse drug resistance in K562 cells resistant to imatinib (IM), the reference drug used in CML therapy. The cytotoxicity of derivative 22 on IM-sensitive and resistant cells was associated with upregulation of FOXN3 and CDKN1A expression, G2/M arrest, and triggering of apoptosis. In conclusion, derivative 22 has chemical-physical characteristics and biological effects superior to CUR, which allow us to hypothesize its future use in the therapy of CML and CML forms resistant to IM, either alone or in combination with this drug.

A Central Contribution of TG2 Activity to the Antiproliferative and Pro-Apoptotic Effects of Caffeic Acid in K562 Cells of Human Chronic Myeloid Leukemia.

Caffeic acid (CA) has shown antitumor activity in numerous solid and blood cancers. We have recently reported that CA is active in reducing proliferation and triggering apoptosis in both Imatinib-sensitive and resistant Chronic Myeloid Leukemia (CML) cells. Tissue transglutaminase type 2 (TG2) enzyme is involved in cell proliferation and apoptosis of numerous types of cancer. However, its activity has different effects depending on the type of tumor. This work investigated the possible involvement of TG2 activation in the triggering of CA-dependent anticancer effects on the K562 cell line, which was studied as a model of CML. CA-dependent changes in TG2 activity were compared with the effects on cell proliferation and apoptosis. The use of N-acetylcysteine (NAC), an antioxidant molecule, suggested that the antiproliferative effect of CA was due to the increase in reactive oxygen species (ROS). The use of a TG2 inhibitor showed that TG2 activity was responsible for the increase in ROS generated by CA and reduced both caspase activation and triggering of CA-dependent apoptosis. The knocking-down of TGM2 transcripts confirmed the crucial involvement of TG2 activation in CML cell death. In conclusion, the data reported, in addition to ascertaining the important role of TG2 activation in the antiproliferative and pro-apoptotic mechanism of CA allowed us to hypothesize a possible therapeutic utility of the molecules capable of triggering the activation pathways of TG2 in the treatment of CML.

Reduction of oxidative stress and ornithine decarboxylase expression in a human prostate cancer cell line PC-3 by a combined treatment with α-tocopherol and naringenin.

Differentiation of a human aggressive PC-3 cancer cell line was obtained, in a previous investigation, by the synergic effect of α-tocopherol (α-TOC) and naringenin (NG). This combined treatment induced apoptosis and subsequent reduction of the PC-3 cell proliferation and invasion, by a pro-differentiating action. Since one of the peculiar characteristics of NG and α-TOC is their strong antioxidant activity, this study aimed to investigate their potential effect on the activity of the main enzymes involved in the antioxidant mechanism in prostate cancer cells. NG and α-TOC administered singularly or combined in the PC-3 cell line, affected the activity of several enzymes biomarkers of the cellular antioxidant activity, as well as the concentration of total glutathione (GSH + GSSG) and thiobarbituric acid reactive substances (TBARS). The combined treatment increased the TBARS levels and superoxide dismutase (SOD) activity, while decreased the glutathione S-transferase (GST), glutathione reductase (GR), and glyoxalase I (GI) activities. The results obtained indicate that a combined treatment with these natural compounds mitigated the oxidative stress in the human PC-3 cell line. In addition, a significant reduction of both ornithine decarboxylase (ODC) expression and intracellular levels of polyamines, both well-known positive regulators of cell proliferation, accompanied the reduction of oxidative stress observed in the combined α-TOC and NG treatment. Considering the established role of polyamines in cell differentiation, the synergism with NG makes α-TOC a potential drug for further study on the differentiation therapy in prostate cancer patients.

Caffeic Acid Enhances the Anti-Leukemic Effect of Imatinib on Chronic Myeloid Leukemia Cells and Triggers Apoptosis in Cells Sensitive and Resistant to Imatinib.

Among the phenolic acids tested on the K562 cell line, a model of chronic myeloid leukemia (CML), caffeic acid (CA) was biologically active on sensitive and imatinib (IM)-resistant cells at micro-molar concentration, either in terms of reduction of cell proliferation or triggering of apoptosis. The CA treatment provoked mitochondrial membrane depolarization, genomic DNA fragmentation and phosphatidylserine exposure, hallmarks of apoptosis. Cell cycle analysis following the treatment with comparable cytotoxic concentrations of IM or CA showed marked differences in the distribution profiles. The reduction of cell proliferation by CA administration was associated with increased expression of two cell cycle repressor genes, CDKN1A and CHES1, while IM at a cytotoxic concentration increased the CHES1 but not the CDKN1A expression. In addition, CA treatment affected the proliferation and triggered the apoptosis in IM-resistant cells. Taken together, these data suggested that CA induced the anti-proliferative effect and triggered apoptosis of CML cells by a different mechanism than IM. Finally, the combined administration of IM and CA at suboptimal concentrations evidenced a synergy of action in determining the anti-proliferative effect and triggering apoptosis. The ability of CA to potentiate the anti-leukemic effect of IM highlighted the nutraceutical potential of CA in CML.

Flavonoids: A Myth or a Reality for Cancer Therapy?

Nutraceuticals are biologically active molecules present in foods; they can have beneficial effects on health, but they are not available in large enough quantities to perform this function. Plant metabolites, such as polyphenols, are widely diffused in the plant kingdom, where they play fundamental roles in plant development and interactions with the environment. Among these, flavonoids are of particular interest as they have significant effects on human health. In vitro and/or in vivo studies described flavonoids as essential nutrients for preventing several diseases. They display broad and promising bioactivities to fight cancer, inflammation, bacterial infections, as well as to reduce the severity of neurodegenerative and cardiovascular diseases or diabetes. Therefore, it is not surprising that interest in flavonoids has sharply increased in recent years. More than 23,000 scientific publications on flavonoids have described the potential anticancer activity of these natural molecules in the last decade. Studies, in vitro and in vivo, show that flavonoids exhibit anticancer properties, and many epidemiological studies confirm that dietary intake of flavonoids leads to a reduced risk of cancer. This review provides a glimpse of the mechanisms of action of flavonoids on cancer cells.

Polyamine Oxidase Is Involved in Spermidine Reduction of Transglutaminase Type 2-Catalyzed ßH-Crystallins Polymerization in Calcium-Induced Experimental Cataract.

In an in vitro Ca2+-induced cataract model, the progression of opacification is paralleled by a rapid decrease of the endogenous levels of spermidine (SPD) and an increase of transglutaminase type 2 (TG2, EC 2.3.2.13)-catalyzed lens crystallins cross-linking by protein-bound N1-N8-bis(γ-glutamyl) SPD. This pattern was reversed adding exogenous SPD to the incubation resulting in a delayed loss of transparency of the rabbit lens. The present report shows evidence on the main incorporation of SPD by the catalytic activity of TG2, toward ßH-crystallins and in particular to the ßB2- and mostly in ßB3-crystallins. The increase of endogenous SPD in the cultured rabbit lens showed the activation of a flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAO EC 1.5.3.11). As it is known that FAD-PAO degrades the N8-terminal reactive portion of N1-mono(γ-glutamyl) SPD, the protein-bound N8-mono(γ-glutamyl) SPD was found the mainly available derivative for the potential formation of ßB3-crystallins cross-links by protein-bound N1-N8-bis(γ-glutamyl)SPD. In conclusion, FAD-PAO degradation of the N8-terminal reactive residue of the crystallins bound N1-mono(γ-glutamyl)SPD together with the increased concentration of exogenous SPD, leading to saturation of glutamine residues on the substrate proteins, drastically reduces N1-N8-bis(γ-glutamyl)SPD crosslinks formation, preventing crystallins polymerization and avoiding rabbit lens opacification. The ability of SPD and MDL 72527 to modulate the activities of TG2 and FAD-PAO involved in the mechanism of lens opacification suggests a potential strategy for the prevention of senile cataract.

Evaluation of polyamines as marker of melanoma cell proliferation and differentiation by an improved high-performance liquid chromatographic method.

The differentiation therapy is focused on the identification of new agents able to impair the proliferative and metastatic potential of cancer cells through the induction of differentiation. Although several markers of cell differentiation on tumor cells have been identified, their causal relationship with neoplastic competence has not been characterized in sufficient detail to propose their use as new pharmacological targets useful for the design of new differentiation agents. Polyamine level in cancer cells and in body fluids was proposed as potential marker of cell proliferation and differentiation. The main advantage of this marker is the possibility to evaluate the antineoplastic activity of new drugs able to induce cell differentiation and consequently to inhibit tumor growth and metastasis. The presented report shows a simply and highly reproducible reverse-phase high-performance liquid chromatographic (HPLC) method for the determination of ortho-phthalaldehyde (OPA) derivatives of polyamines: putrescine (PUT), cadaverine (CAD), spermidine (SPD) and spermine (SPM). The novelty of this method is the fluorescence response for OPA-derivate of SPM, generally low in other procedures, that has been significantly improved by the use of a fully endcapped packing material with minimal silanol interactions. The limits of detection for PUT, CAD, SPD and SPM were 0.6, 0.7, 0.8, and 0.4 pmol/mL, respectively. The analysis time was ≤ 20 min, and the relative recovery rate was of about 97%. To verify the usefulness of this method, it has been validated in a murine melanoma cell line (B16-F10) treated with two theophylline derivatives (namely 8-chlorotheophylline and 8-bromotheophylline). These two compounds increased the activity of tissue transglutaminase (TG2) and the synthesis of melanin, two recognized markers of melanoma cell differentiation, and significantly reduced the levels of intracellular polyamines.

Chemical Composition of Essential Oils from Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis, and Their Effects on the HIV-1 Tat Protein Function.

New drugs would be beneficial to fight resistant HIV strains, in particular those capable of interfering with essential viral functions other than those targeted by highly active antiretroviral therapy drugs. Despite the central role played by Tat protein in HIV transcription, a search for vegetable extracts able to hamper this important viral function was never carried out. In this work, we evaluated the chemical composition and possible interference of essential oil from Thymus vulgaris, Cananga odorata, Cymbopogon citratus, and Rosmarinus officinalis with the Tat/TAR-RNA interaction and with Tat-induced HIV-1 LTR transcription. GC/MS Analysis demonstrated the biodiversity of herbal species translated into essential oils composed of different blends of terpenes. In all of them, 4 - 6 constituents represent from 81.63% to 95.19% of the total terpenes. Essential oils of Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis were active in interfering with Tat functions, encouraging further studies to identify single terpenes responsible for the antiviral activity. In view of the quite different composition of these essential oils, we concluded that their interference on Tat function depends on specific terpene or a characteristic blend.

Characterization of Stable Pyrazole Derivatives of Curcumin with Improved Cytotoxicity on Osteosarcoma Cell Lines.

Curcumin (CUR) is a natural molecule that is unstable due to the presence of a bis-ketone. To obtain more stable derivatives in biological fluids, the bis-ketone was replaced with pyrazole or O-substituted oximes. Their stability in solution was studied by UV-visible spectrophotometry. The effects on proliferation were studied by MTT assay and/or clonogenicity assay. Induction of apoptosis was evaluated by annexin V staining and Western blot analysis. The bioavailability was obtained from the analysis of the molecular chemical-physical characteristics. The replacement of the bis-ketone with a pyrazole ring or O-substituted oximes improved the stability of all the CUR-derivative molecules. These derivatives were more stable than CUR in solution and were generally cytotoxic on a panel of cancer cell lines tested, and they promoted caspase-dependent apoptosis. Derivative 1 was the most potent in the osteosarcoma (OS) lines. With respect to CUR, this derivative showed cytotoxicity at least three times higher in the MTT assay. In addition, in the clonogenic assay, 1 maintained the activity in conditions of long treatment presumably by virtue of its improved stability in biological fluids. Notably, 1 should have improved chemical-physical characteristics of bioavailability with respect to CUR, which should allow for reaching higher blood levels than those observed in the CUR trials. In conclusion, 1 should be considered in future clinical studies on the treatment of OS, either alone or in combination with other medications currently in use.

Reduction in Fatigue Symptoms Following the Administration of Nutritional Supplements in Patients with Multiple Sclerosis.

Despite recent advances in immune-modulatory drugs, pharmacological therapies have been proven ineffective in severe presentations of multiple sclerosis (MS), including secondary progressive MS. At present, therapeutic interventions' performance is primarily focused on ameliorating symptoms to improve the patient's quality of life (QOL). Among complementary treatments, nutrition has been considered a decisive factor to control symptoms and enhance the wellness of MS patients. Although no special diets are associated with MS, the impact of diet and dietary supplements on the course of progressive forms of the disease has been studied during the last few years. Fatigue is among the most common and disabling symptoms reported by MS patients. Fatigue has been defined in the Multiple Sclerosis Council for Clinical Practice Guidelines (MSCCPG, 1998) as a "subjective lack of physical and/or mental energy that the individual perceives as an interference with habitual and desired activities". This study aimed to compare the psychometric functioning of the "Fatigue Severity Scale" (FSS) and the "Modified Fatigue Impact Scale" (MFIS) in our sample of people with MS. Specifically, during chronic treatment, the change in these two parameters with two vitamin-rich dietary supplements (Citozym® and Ergozym®) was evaluated. The impact of these nutritional supplements revealed differences in antioxidant and anti-inflammatory parameters among the volunteers in the treatment group, with a subsequent improvement in fatigue. In conclusion, the results obtained have confirmed the effectiveness of complementary nutritional therapies, evaluated essentially based on hematological biomarkers, through which it is possible to act on disability to improve the QOL of MS patients.

Production of beta-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous beta(0)39 thalassemia patients.

In several types of thalassemia (including beta(0)39-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying beta-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the beta(0)39-thalassemia globin gene under control of the beta-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of beta-globin by K562 cell clones expressing the beta(0)39-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from beta(0)39-thalassemia patients were demonstrated to be able to produce beta-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of beta(0)-thalassemia caused by stop codon mutations. Am. J. Hematol., 2009. (c) 2009 Wiley-Liss, Inc.

Development of K562 cell clones expressing beta-globin mRNA carrying the beta039 thalassaemia mutation for the screening of correctors of stop-codon mutations.

Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in beta(0)39-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon-anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of beta(0)39-thalassaemia. In this context, we started the development of a cellular model of the beta(0)39-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the beta(0)39-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of beta-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the beta(0)39-globin mutation causing beta-thalassaemia.

The Role of Tissue Transglutaminase in Cancer Cell Initiation, Survival and Progression.

Tissue transglutaminase (transglutaminase type 2; TG2) is the most ubiquitously expressed member of the transglutaminase family (EC 2.3.2.13) that catalyzes specific post-translational modifications of proteins through a calcium-dependent acyl-transfer reaction (transamidation). In addition, this enzyme displays multiple additional enzymatic activities, such as guanine nucleotide binding and hydrolysis, protein kinase, disulfide isomerase activities, and is involved in cell adhesion. Transglutaminase 2 has been reported as one of key enzymes that is involved in all stages of carcinogenesis; the molecular mechanisms of action and physiopathological effects depend on its expression or activities, cellular localization, and specific cancer model. Since it has been reported as both a potential tumor suppressor and a tumor-promoting factor, the role of this enzyme in cancer is still controversial. Indeed, TG2 overexpression has been frequently associated with cancer stem cells' survival, inflammation, metastatic spread, and drug resistance. On the other hand, the use of inducers of TG2 transamidating activity seems to inhibit tumor cell plasticity and invasion. This review covers the extensive and rapidly growing field of the role of TG2 in cancer stem cells survival and epithelial⁻mesenchymal transition, apoptosis and differentiation, and formation of aggressive metastatic phenotypes.

Upstream stimulatory factors are involved in the P1 promoter directed transcription of the A beta H-J-J locus.

BACKGROUND: Alternative splicing of the locus A beta H-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) beta-hydroxylase (AAH), truncated homologs of AAH with a role in calcium homeostasis humbug and junctate and a structural protein of the sarcoplasmic reticulum membranes junctin. AAH and humbug are over expressed in a broad range of malignant neoplasms. We have previously reported that this locus contains two promoters, P1 and P2. While AAH and humbug are expressed in most tissues under the regulation of the P1 promoter, AAH, junctin and junctate are predominantly expressed in excitable tissues under the control of the P2 promoter. We previously demonstrated that Sp transcription factors positively regulate the P1 promoter. RESULTS: In the present study, we extended the functional characterization of the P1 promoter of the A beta H-J-J locus. We demonstrated by quantitative Real-time RT-PCR that mRNAs from the P1 promoter are actively transcribed in all the human cell lines analysed. To investigate the transcription mechanism we transiently transfected HeLa cells with sequentially deleted reporter constructs containing different regions of the -661/+81 P1 nucleotide sequence. Our results showed that (i) this promoter fragment is a powerful activator of the reporter gene in HeLa cell line, (ii) the region spanning 512 bp upstream of the transcription start site exhibits maximal level of transcriptional activity, (iii) progressive deletions from -512 gradually reduce reporter expression. The region responsible for maximal transcription contains an E-box site; we characterized the molecular interactions between USF1/2 with this E-box element by electrophoretic mobility shift assay and supershift analysis. In addition, our USF1 and USF2 chromatin immunoprecipitation results demonstrate that these transcription factors bind the P1 promoter in vivo. A functional role of USF1/USF2 in upregulating P1-directed transcription was demonstrated by analysis of the effects of (i) in vitro mutagenesis of the P1/E-box binding site, (ii) RNA interference targeting USF1 transcripts. CONCLUSION: Our results suggest that USF factors positively regulate the core of P1 promoter, and, together with our previously data, we can conclude that both Sp and USF DNA interaction and transcription activity are involved in the P1 promoter dependent expression of AAH and humbug.

Myocyte enhancer factor 2 activates promoter sequences of the human AbetaH-J-J locus, encoding aspartyl-beta-hydroxylase, junctin, and junctate.

Alternative splicing of the locus AbetaH-J-J generates three functionally distinct proteins: an enzyme, AbetaH (aspartyl-beta-hydroxylase), a structural protein of the sarcoplasmic reticulum membrane (junctin), and an integral membrane calcium binding protein (junctate). Junctin and junctate are two important proteins involved in calcium regulation in eukaryotic cells. To understand the regulation of these two proteins, we identified and functionally characterized one of the two promoter sequences of the AbetaH-J-J locus. We demonstrate that the P2 promoter of the AbetaH-J-J locus contains (i) a minimal sequence localized within a region -159 bp from the transcription initiation site, which is sufficient to activate transcription of both mRNAs; (ii) sequences which bind known transcriptional factors such as those belonging to the myocyte enhancer factor 2 (MEF-2), MEF-3, and NF-kappaB protein families; and (iii) sequences bound by unknown proteins. The functional characterization of the minimal promoter in C2C12 cells and in the rat soleus muscle in vivo model indicates the existence of cis elements having positive and negative effects on transcription. In addition, our data demonstrate that in striated muscle cells the calcium-dependent transcription factor MEF-2 is crucial for the transcription activity directed by the P2 promoter. The transcription directed by the AbetaH-J-J P2 promoter is induced by high expression of MEF-2, further stimulated by calcineurin and Ca2+/calmodulin-dependent protein kinase I, and inhibited by histone deacetylase 4.

New trends in non-invasive prenatal diagnosis: applications of dielectrophoresis-based Lab-on-a-chip platforms to the identification and manipulation of rare cells.

The isolation of rare cells, such as fetal nucleated red blood cells and trophoblasts, from maternal blood for non-invasive prenatal diagnosis is a new field of research exhibiting several difficulties since this strategy requires unresolved basic technological protocols for a successful outcome. However, several achievements in the field of Laboratory-on-a-chip (Lab-on-a-chip) technology have provided clear advancements in projects aimed at the isolation of rare cells from biological fluids. Among the most interesting approaches are those based on dielectrophoresis (DEP). DEP-based Lab-on-a-chip platforms have been demonstrated to be suitable for several applications in biotechnology and biomedicine. DEP-based arrays are able to manipulate single cells, which can be identified and moved throughout the DEP chip to recovery places. DEP buffers are compatible with molecular interactions between monoclonal antibodies and target cells, allowing integration of these devices with magnetic cell sorting (MACS). DEP treatment does not alter the viability of manipulated cells.

A novel frameshift mutation (+A) at codon 18 of the beta-globin gene associated with high persistence of fetal hemoglobin phenotype and deltabeta-thalassemia.

We report in this paper a novel thalassemia mutation (insertion of a single A nucleotide within the exon 1, at codon 18, of the beta-globin gene) associated with a deletion of the deltabeta-globin gene region, in a patient exhibiting high persistence of fetal hemoglobin. The novel mutation causes a frameshift with the generation of a UGA stop codon. Analysis of the parent's DNA demonstrates that the A insertion and frameshift mutation are inherited from the father, while the deltabeta-globin gene deletion is inherited from the mother. Gene dosage analysis and deletion-specific PCR demonstrate that the deletion is the (deltabeta)(0) Sicilian deletion, involving a 13.4-kb deltabeta-globin gene region.

Selected terpenes from leaves of Ocimum basilicum L. induce hemoglobin accumulation in human K562 cells.

Re-expression of fetal hemoglobin (HbF) was proposed as a possible therapeutic strategy for ß-haemoglobinopathies. Although several inducers of HbF were tested in clinical trials, only hydroxyurea (HU) received FDA approval. Despite it produced adequate HbF levels only in half of HU-treated SCD patients, and was ineffective at all in ß-thalassemia patients, beneficial effects of this approach suggested to continue in this direction identifying further molecules capable of inducing HbF. We tested the potential of essential oil isolated from Ocimum basilicum L. leaves (ObEO) in inducing hemoglobin biosynthesis. Initially, dose-dependent effect and kinetics of hemoglobin accumulation in K562 cells after treatment with ObEO were evaluated. ObEO induced dose-dependent hemoglobin accumulation superior to hydroxyurea and rapamycin and a strongest γ-globin mRNA expression. Terpenes composition of ObEO was studied by GC-MS. Three main constituents, linalool, eugenol and eucalyptol, represented about 75% of total. A blend of these three terpenes fully replicated the ObEO's biological effect, thus indicating that one of them or all together could be the active ingredients. When terpenes were tested individually, eugenol was the only one inducing stable hemoglobin accumulation, while eucalyptol and linalool produced only a small transient response. However, eugenol potential was strongly enhanced in the presence of eucalyptol and linalool, suggesting a synergistic effect on hemoglobin accumulation. By these results, the discovery of a new inducer and the interesting activity of a blend of major terpenes from ObOE on Hb accumulation could have positive fallouts on ß-thalassemia and sickle cells anemia.

Antiproliferative activity of novel isatinyl/indanyl nitrones (INs) as potential spin trapping agents of free radical intermediates.

A series of ketonitrones derived from isatin and indanone (INs) were synthesized and evaluated for their antiproliferative activities against several human cancer cell lines. Then, the antioxidant properties of these substrates were measured by the DPPH test to report their biological activity in terms of their spin trapping action. In particular, one substrate has showed very high biological and scavenging activity, probably due to the strong correlation between its spin trapping activity and structure.