shRNA targeting of ferritin heavy chain activates H19/miR-675 axis in K562 cells.

PURPOSE: The heavy subunit of the iron storage protein ferritin (FHC) is essential for the intracellular iron metabolism and, at the same time, it represents a central hub of iron-independent pathways, such as cell proliferation, angiogenesis, p53 regulation, chemokine signalling, stem cell expansion, miRNAs expression. In this work we have explored the ability of FHC to modulate gene expression in K562 cells, through the up-regulation of the lncRNA H19 and its cognate miR-675. MATERIALS AND METHODS: Targeted silencing of FHC was performed by lentiviral-driven shRNA strategy. FHC reconstitution was obtained by full length FHC cDNA transfection with Lipofectamine 2000. ROS amounts were determined with the redox-sensitive probe H2DCFDA. H19, miR-675, miR-107, Twist1, ID3, EPHB6, GNS, ANK1 and SMAD6 mRNA amounts were quantified by Taqman assay and qPCR analysis. RESULTS: FHC silencing in K562 cells modulates gene expression through the up-regulation of the lncRNA H19 and its cognate miR-675. Experimental findings demonstrate that the molecular mechanism underlying this phenomenon is represented by an FHC knock-down-triggered increase in reactive oxygen species (ROS) production. CONCLUSIONS: In this paper we uncover a so far not described function of the ferritin heavy subunit in the control of lncRNA pathways.

Sgk1 enhances RANBP1 transcript levels and decreases taxol sensitivity in RKO colon carcinoma cells.

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of epithelial sodium channel-mediated sodium transport and is involved in the transduction of growth factor-dependent cell survival and proliferation signals. Growing evidence now points to Sgk1 as a key element in the development and/or progression of human cancer. To gain insight into the mechanisms through which Sgk1 regulates cell proliferation, we adopted a proteomic approach to identify up- or downregulated proteins after Sgk1-specific RNA silencing. Among several proteins, the abundance of which was found to be up- or downregulated upon Sgk1 silencing, we focused our attention of RAN-binding protein 1 (RANBP1), a major effector of the GTPase RAN. We report that Sgk1-dependent regulation of RANBP1 has functional consequences on both mitotic microtubule activity and taxol sensitivity of cancer cells.

Negative transcriptional regulation of the human periostin gene by YingYang-1 transcription factor.

Periostin (POSTN), an osteoblast-specific secreted protein known to be associated with cell adhesion activity for bone formation and development by the epithelial cell-derived tumors, leads to a significant enhancement in angiogenesis and tumorigenesis. At present, little is known about the mechanisms underlying its transcriptional control either in physiological or neoplastic conditions. In this study we demonstrate that the ability of the human POSTN promoter to drive transcription mostly depends on the activity of YingYang-1 (YY1) zinc finger transcription factor. YY1, whose regulatory role in biology includes, besides transcriptional control, also chromatin remodeling, DNA damage repair and tumorigenesis, acts as a strong negative modulator of the POSTN expression. We retain that the identification of the functional role of YY1 in the transcriptional control of the human POSTN gene adds new insights in the studies focused on gene expression in normal and transformed cells.

Common variable immunodeficiency in vitiligo.

Three cases of adult, non-segmental vitiligo patients in which primary, common variable immunodeficiency (CVID) was present are described. In two of the three cases, psoriasis was also present. Onset of CVID was diagnosed before vitiligo in two patients, and subsequent to the onset of vitiligo in the remaining patient. A family history of CVID was negative in all three cases. In contrast, a family history of vitiligo was present in two cases and a family history of psoriasis was present in one case. In regards to vitiligo, disease onset was gradual, with active disease in two cases, while the third case had an abrupt disease onset with borderline activity during clinical presentation. Cutaneous disease extension ranged from 2% to 12.3% in the three cases. Upon physical exam, Koebner phenomenon and signs of inflammation, such as pruritus were present in two patients; one of whom had scalp leukotrikia as well. Stress was a triggering factor in the development of vitiligo in two cases. The presence of CVID due to drugs or diseases known to cause secondary antibody deficiency were excluded in all three patients. Lastly, all patients were under treatment with immunoglobulin replacement therapy, which did not change the outcourse of vitiligo.

Stress as a possible mechanism in melanoma progression.

The incidence of melanoma, the most aggressive type of cutaneous malignant tumor, is currently on the rise. Treatment in advanced stages is still unsuccessful compared with other malignant tumors, thus it is important to indentify the key mechanisms responsible for melanoma progression and metastasis. Genetic and molecular components, in particular, that are up- or downregulated in melanoma cells, affect the invasive potential of melanoma. Another possible important cofactor highlighted by recent studies is chronic stress, involving environmental and psychological factors, which can be an important cofactor in not only cancer progression in general but also in melanoma spreading. The negative effects of chronic stress have been evaluated epidemiologically in patients with breast and prostate cancer. In particular, the effects of stress mediators, namely, catecholamines have been studied on various human malignancies, including melanoma and have highlighted a significant increase of progression-related molecules. As such, this could be the starting point for a new approach in the treatment of advanced melanoma, in which the negative effects of stress are reduced or blocked.

O6-Methylguanine-DNA-methyltransferase in recurring anaplastic ependymomas: PCR and immunohistochemistry.

Ependymomas are the third most common brain tumor in children. The post surgical management is controversial. There are no convincing data on an effective role for chemotherapy. O(6)-Methylguanine-DNA-Methyltransferase (MGMT) is a DNA repair protein considered to be a chemosensitivity predictor. Hypermethylation of the MGMT gene promoter is an important cause of MGMT inactivation. We evaluated the MGMT gene promoter methylation and the immunohistochemical MGMT protein expression in 12 recurrent anaplastic ependymomas affecting children. Our purpose was to investigate the molecular rationale of the administration of alkylating agents to children affected by recurrent anaplastic ependymomas. All ependymomas lacked MGMT promoter hypermethylation and 9 (75%) showed high MGMT protein expression (>50% tumoral cells). Differences between different recurrences in the same patient were not observed. These results may indicate MGMT as a factor of chemoresistance to alkylating drugs in anaplastic ependymomas and support the uncertainties regarding the actual benefit of chemotherapy for patients with anaplastic ependymomas.

Occipital pilomyxoid astrocytoma in a 14-year-old girl--case report.

Pilomyxoid astrocytoma is a recently described tumor. Its most typical morphological characteristics are an angiocentric astrocytic proliferation embedded in a myxoid background. The behavior seems to be unfavorable due to the reported high rate of local recurrence. The earlier studies indicated that pilomyxoid astrocytoma typically affects young children and arises in the hypothalamic/chiasmatic region. We report a case of a 14-year-old patient with a 6-year history of absence seizure. Magnetic resonance imaging showed a right occipital lesion of approximately 3 cm in diameter. The patient underwent the surgical procedure with gross total excision. Histologically, the tumor was mainly composed of a monomorphous population of bipolar elongated piloid cells radially arranged around thin-walled blood vessels in a prominent myxoid background. There were focal hemorrhagic foci but no bona fide evidence of tumor necrosis or mitoses. Rosenthal fibers and eosinophilic granular bodies were not observed. The postoperative course was uneventful. No adjuvant therapy was administered. The patient is alive and well at 18-month follow-up. The case presented provides evidence that pilomyxoid astrocytoma can occur at a later age and can arise in regions different from hypothalamic/chiasmatic.

Giant cyst of the cavum septi pellucidi, cavum Vergae and veli interpositi.

The authors report a case study of a giant cyst of the cavum septi pellucidi, cavum Vergae and veli interpositi spreading to the posterior fossa, and initially treated elsewhere by ventriculoperitoneal shunt, with no resolution of the symptomatology. A few months later the patient was successfully treated by fenestration into the ventricular system through a neuroendoscopic technique, at the Pediatric Neurosurgical Center of the Meyer Children's Hospital in Florence. Symptomatic midline cysts are quite rare and different techniques have been proposed for their treatment, i. e., direct craniotomy, conventional shunting, stereotactic approaches as well as endoscopic fenestration. In such cases neuroendoscopy obtains a good symptom resolution level, avoiding at the same time the risks of damage to endoventricular structures and often eliminates the need for a definitive ventriculoperitoneal shunt. In the present research the authors analyze the anatomy of the midline cavities and the mechanism through which a cyst may become symptomatic. The surgical endoscopic technique and the clinical and radiological assessments which confirmed the patency of the fenestration are also discussed. The authors conclude that endoscopic ventricular fenestration may represent the treatment of choice for this pathology.

Manejo y consumo de productos dietéticos y edulcorantes no nutritivos / Handling and consumption of dietetic products and no nutritional

Habiendo analizado en estudiantes de nutrición (1998) el consumo de edulcorantes no nutritivos (ENN), productos que lo contengan y el uso de los mismos por la Industria, se continúa con la línea de investigación, analizando otras "poblaciones riesgo" (PR).Objetivos: Determinar en el término de un año (98/99) la variación en la Industria de productos dietéticos (PD) con ENN. Establecer su manejo y prevalencia de consumo en PR y porcentaje de adecuación en relación a la ingesta diaria admisible (IDA) para cada uno. Metodología: Se estudió como ENN la Sacarina (S), Ciclamato©, Acesulfame K (Ac), Aspartamo (A) y Sucralosa (Su). Población n=290 sexo femenino. Se consideró PR a la expuesta al mayor consumo de PD y ENN, ya sea por situación fisiopatológica presente, período biológico o influencias de medios de comunicación. Se establecieron 3 grupos: adolescentes n=80; adultas en edad fértil n=120 y perimenopáusicas n=90.Resultados: El uso por la Industria de PD y ENN aumentó en un año un 49,1 por ciento incorporándose la Su y aumentando la utilización de Ac:100,0 por ciento, A:28,6 por ciento, C: 25,7 por ciento y S:12,5 por ciento. El rubro de PD que más creció fue el de yogures (157,1 por ciento).Con respecto al manejo de PD y ENN, en las 3 poblaciones estudiadas, la mayoría "se cuida" o no realiza ninguna alimentación especial; utilizan como criterio para seleccionar a los PD el sabor de los mismos; leen el rotulado nutricional, principalmente el aporte calórico y los ENN son seleccionados también por el sabor. Los PD más consumidos son chicles, mermeladas, jugos, gelatinas, gaseosas, yogures, edulcorantes, frutas enlatadas, flanes y postres de leche y alfajores. No hubo variaciones en las marcas consumidas con el estudio anterior. Con respecto al porcentaje de adecuación para la IDA, la mayoría (90 por ciento) se encuentra por debajo del 25 por ciento de adecuación para S, C, A y Ac. Ninguno supera el 100 por ciento de adecuación. No se pudo determinar la adecuación para la Su, por no declarar el rotulado la cantidad contenida en los productos. Conclusiones: Pese a la mayor disponibilidad y aumento del uso por la industria de PD con ENN, se observa en el consumo que el porcentaje de adecuación para la IDA de cada uno de ellos, es menor al 25 por ciento (AU)

[Local thrombolysis in renal artery embolism]. / Fibrinolisis local en el embolismo de la arteria renal.

Renal artery embolism is an infrequent entity that occurs in patients with underlying cardiac diseases. Diagnosis is usually difficult unless the index of suspicion is high. Local thrombolysis with low-dose fibrinolytic agents is an useful therapeutic intervention. We present 2 cases of renal artery embolism treated with intra-arterial urokinase and review clinical features and therapeutic options.

Fibrinolisis local en el embolismo de la arteria renal / Local thrombolysis in renal artery embolism

El embolismo de la arteria renal es una entidad rara que se presenta en pacientes con cardiopatías embolígenas y que comporta dificultades diagnósticas si no se mantiene un índice de sospecha muy elevado. La utilización de agentes fibrinolíticos locales constituye una opción terapéutica útil. Presentamos dos casos de embolismo de la arteria renal tratados con urokinasa intraarterial y revisamos las manifestaciones clínicas y las alternativas terapéuticas existentes (AU)

Effect of peptides on the inactivation of tissue plasminogen activator by plasminogen activator inhibitor-1 and on the binding of tissue plasminogen activator to endothelial cells.

The effectiveness of tissue plasminogen activator (tPA) in thrombolytic therapy is dependent upon the rate at which therapeutically administered tPA reaches the clot site and the proportion of that tPA which is enzymatically active. Interactions between tPA and its main plasma inhibitor (PAI-1) and between tPA and the endothelial cells lining blood vessels are two factors which may limit efficacy. In an attempt to identify the regions of the tPA molecule involved in these interactions, we have examined a series of synthetic peptides with amino acid sequences corresponding to different regions of the tPA molecule for their ability to protect tPA from inactivation by PAI-1 and for their ability to reduce the binding of tPA to endothelial cells. Three peptides were identified which were especially effective at maintaining tPA activity in the presence of PAI-1 and three others were found which had a lesser effect. These same peptides were also found to inhibit the binding of tPA to endothelial cells. This suggests that the same regions of the tPA molecule are involved in both processes. None of the peptides inhibited the binding of tPA to fibrin. These peptides may serve as models for the development of agents for enhancing the activity of both endogenous tPA and of tPA administered in thrombolytic therapy.

The use of competitive affinity chromatography for the isolation of proteins which promote transcription.

A method is described for isolating proteins which bind preferentially to specific sequences of DNA and is used to enrich preparations in proteins promoting eukaryotic gene transcription. An ion-exchange fraction which promotes the transcription of the ovalbumin gene in in vitro runoff assays was isolated from the oviducts of diethylstilbesterol-stimulated chicks. This fraction was recirculated between two coupled columns, one containing random sequences of prokaryotic DNA and the other a specific cloned DNA fragment from the 5' region of the ovalbumin gene. Recirculation was performed in the presence of a decreasing salt gradient, after which columns were disconnected and separately eluted. The eluate obtained from the column containing cloned DNA showed a preference for binding to DNA fragments derived from the ovalbumin gene when examined in nitrocellulose filter binding assays. This fraction retained the ability of its precursor to promote gene transcription in a concentration-dependent manner. Active preparations were also examined in assays measuring total RNA synthesis from native DNA templates. Although the fraction isolated from the column of specific DNA had no detectable RNA polymerase activity itself, it enhanced the activity of calf thymus RNA polymerase II. The method presented should find general application in the purification of factors which regulate biological processes by binding to specific sequences of DNA.

Binding of tissue plasminogen activator to human aortic endothelial cells.

The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.

Purification and characterization of a tissue plasminogen activator-inhibitor complex from human umbilical vein endothelial cell conditioned medium.

Tissue plasminogen activator-inhibitor complexes were purified from the conditioned medium of human umbilical vein endothelial cells by affinity chromatography followed by gel filtration. It was found that a single complex was isolated which can exist in two distinct interconvertible conformations. These may be separated by electrophoresis into a form with a 105,000 apparent molecular weight and a form with an 88,000 apparent molecular weight. The particular conformation which predominates may be altered by changing the pH at which preparations are incubated or by including dithiothreitol in incubation buffers. Plasminogen activator enzymatic activity may be partially recovered from purified complexes by incubation in the presence of fibrin. Incubation in 1.5 M NH4OH results in the dissociation of the complex into two major polypeptides of 67 and 40 kilodaltons (kDa). The 40-kDa protein was isolated by gel filtration high-pressure liquid chromatography. N-Terminal amino acid analysis of this protein revealed three distinct sequences. Two of these were nearly identical and matched the N-terminal sequence recently reported for the native plasminogen activator inhibitor from endothelial cells. The third sequence exactly matched an internal portion of the same protein. The results suggest that the internal sequence is located at the site where the inhibitor is cleaved by tissue plasminogen activator.

Isolation of a protein fraction that binds preferentially to chicken middle repetitive DNA.

We have fractionated oviduct tissue extracts by using a combination of ion-exchange and DNA-Sephadex chromatography. By comparing the electrophoretic patterns of proteins eluted from competing specific and nonspecific DNA columns, we isolated a fraction which bound with specificity to columns containing the chicken middle repetitive sequence "CR1". This fraction showed a clear preference for binding to separate, cloned CR1 fragments derived from either the 5' or the 3' transition region of the ovalbumin gene domain when examined by using nitrocellulose filter binding assays. To localize the protein binding site, a CR1 clone was digested with various restriction enzymes, and the resulting fragments were examined for preferential protein binding. Results suggest that the binding site lies within a 39-nucleotide sequence which is highly conserved among different CR1 elements. This finding represents the first isolation of a protein which demonstrates a preference for binding to a middle repetitive sequence and suggests that this interaction may have a biological role. The DNA column competition adsorption method should have general application to the isolation of other gene-regulating proteins possessing DNA sequence preference.

Gonadotropin receptor binding regulators in serum. Characterization and separation of follitropin binding inhibitor and lutropin binding stimulator.

Subfractions of human serum capable of inhibiting specific binding of radioiodinated human follitropin and stimulating specific binding of radioiodinated human lutropin to testicular receptors were prepared by molecular sieving techniques, separated by ion exchange chromatography, and further purified by reversed phase high pressure liquid chromatography. Although purified approximately 2000-fold, the exact chemical nature of the follicle-stimulating hormone binding inhibitor is as yet uncertain. However, a variety of analytical procedures indicated it to be separate and distinct from the lutropin binding stimulator, to have a molecular weight of about 700, and to contain a peptide component. Available evidence suggests that the serum factor responsible for luteinizing hormone binding stimulation may be calcium.

Inhibition of FSH action on granulosa cells by low molecular weight components of follicular fluid.

Immature female rats were injected with a single dose (10 IU) of pregnant mare serum gonadotropin to induce growth and maturation of ovarian follicles. Using such ovaries as a model, we tested the effects of low molecular weight subfractions of charcoal-absorbed bovine follicular fluid (FF-c) on (a) radioiodinated human FSH (125I-hFSH) binding to ovarian homogenates, (b) ovine FSH-stimulated adenylate cyclase activity in granulosa cell homogenates and (c) cAMP production by intact granulosa cells. The follicular fluid was fractionated by ultrafiltration through membranes of differing pore-sized into molecular weight components of 1000-5000 (passing Amicon H1P-5 hollow fibers but retained by Amicon UM-2 membrane) and 500-1000 (passing Amicon UM-2 membrane but retained by Amicon Um-05 membrane). These low molecular weight fractions inhibited 125I-hFSH binding to ovarian receptors, FSH-stimulated cAMP production by rat granulosa cells and FSH-stimulated, as well as fluoride-ion-stimulated adenylate cyclase activity in granulosa cell homogenates. Inhibition of FSH-stimulated adenylate cyclase activity by the FF subfractions was non-competitive as determined by double reciprocal plot analysis. Our results suggest that modulation of FSH effects on granulosa cells may be mediated by low molecular weight constituents of follicular fluid.