Descripción: Fil:Crámer, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Descripción: 1. 1. Porphyrin biosynthesis from 5-aminoevulinic acid (ALA) was investigated using the technique of tissue explant cultures, in both human breast cancer and its original normal tissue. 2. 2. The activity of ALA-dehydratase, porphobilinogenase and uroporphyrinogen decarboxylase was directly determined in both tumor and normal mammary tissues. 3. 3. Porphyrin synthesis capacity of human breast carcinoma was 20-fold enhanced, as compared with normal tissue, at least between the stages of porphobilinogen and coproporphyrinogen formation. 4. 4. The activity of the three enzymes examined was always lower in normal tissue than in tumoral tissue. 5. 5. Present findings show that porphyrin biosynthesis is increased in breast cancer tissue. © 1990.

Descripción: The G protein-coupled receptor oncogene (vGPCR) of the Kaposi's sarcoma (KS) associated herpesvirus (KSHV), an oncovirus implicated in angioproliferative neoplasms, induces angiogenesis by VEGF secretion. Accordingly, we found that expression of vGPCR in human umbilical vein endothelial cells (HUVEC) leads to immortalization with constitutive VEGF receptor-2/ KDR expression and activation. vGPCR immortalization was associated with anti-senescence mediated by alternative lengthening of telomeres and an anti-apoptotic response mediated by vGPCR constitutive signaling and KDR autocrine signaling leading to activation of the PI3K/AKT pathway. In the presence of the KS growth factor VEGF, this mechanism can sustain suppression of signaling by the immortalizing gene. We conclude that vGPCR can cause an oncogenic immortalizing event and recapitulate aspects of the KS angiogenic phenotype in human endothelial cells, pointing to this gene as a pathogenic determinant of KSHV.

Descripción: 1. 1. A method for purifying human erythrocytes ALA-D, using a mixture of n-butanol and chloroform, which denature hemoglobin, followed by ammonium sulphate fractionation and affinity chromatography yielding a 1600-fold purified enzyme, is described. 2. 2. By oxidation of Sephadex G-25 with NaIO4, a polyaldehyde, is obtained which can be covalently bound to the ALA-D; however the immobilized enzyme is inactive, because essential ε{lunate}-amino groups at the active site were involved in the coupling. Similar experiments with another enzyme, Rhodanese, resulted in an active insolubilized preparation. 3. 3. By suspending the carrier-enzyme in buffer, slow solubilization with simultaneous release of protein occurs, indicating that this approach might find important therapeutical applications in the treatment of enzyme deficiencies. © 1980.

Descripción: In this work we present a method to detect and recognize the signs of the card game of Truco which are a subset of facial gestures. The method uses temporal templates to represent motion and later extract features. The proposed method works in real time, allowing to use it as an human-computer interface , for example, in the context of the card game of Truco . To the best of our knowledge this is the first work that uses detection of facial gestures in the context of a game. © 2012 Springer-Verlag.

Descripción: Heme oxygenase-1 (HO-1), an inducible enzyme that metabolizes the heme group, is highly expressed in human Kaposi sarcoma lesions. Its expression is up-regulated by the G protein-coupled receptor from the Kaposi sarcoma-associated herpes virus (vGPCR). Although recent evidence shows that HO-1 contributes to vGPCR-induced tumorigenesis and vascular endothelial growth factor (VEGF) expression, the molecular steps that link vGPCR to HO-1 remain unknown. Here we show that vGPCR induces HO-1 expression and transformation through the Gα12/13 family of heterotrimeric G proteins and the small GTPase RhoA. Targeted small hairpin RNA knockdown expression of Gα12, Gα13, or RhoA and inhibition of RhoA activity impair vGPCR-induced transformation and ho-1 promoter activity. Knockdown expression of RhoA also reduces vGPCR-induced VEFG-A secretion and blocks tumor growth in a murine allograft tumor model. NIH-3T3 cells expressing constitutively activated Gα13 or RhoA implanted in nude mice develop tumors displaying spindle-shaped cells that express HO-1 and VEGF-A, similarly to vGPCR-derived tumors. RhoAQL-induced tumor growth is reduced 80% by small hairpin RNA-mediated knockdown expression of HO-1 in the implanted cells. Likewise, inhibition of HO-1 activity by chronic administration of the HO-1 inhibitor tin protoporphyrin IX to mice reduces RhoAQL-induced tumor growth by 70%. Our study shows that vGPCR induces HO-1 expression through the Gα12/13/RhoA axes and shows for the first time a potential role for HO-1 as a therapeutic target in tumors where RhoA has oncogenic activity.

Descripción: Background: Human spermatozoa decondense in vitro upon exposure to heparin and glutathione. Glutathione is also the disulfide bond reducer in vivo, and heparan sulfate, a functional analogue of heparin, has been proposed as the protamine acceptor. The aim of this study was to evaluate the decondensing ability of chemically modified heparins and different glycosaminoglycans (GAGs) on isolated sperm nuclei in vitro, and to analyse the possible role of different GAGs as protamine acceptors. Methods: Capacitated spermatozoa and isolated sperm nuclei from normospermic semen samples were decondensed in the presence of heparin (or its equivalent) and glutathione. After fixation with glutaraldehyde, the percentage of decondensed spermatozoa and nuclei was determined under phase-contrast. Proteins were extracted from sperm nuclei previously incubated in the presence of gluhathione and different GAGs by incubation with urea-β-meracptoethanol-NaCl, and analysed by acid polyacrylamide gel electrophoresis. Results: The ability of desulfated heparins and other GAGs to decondense isolated nuclei mirrored exactly the decondensation of capacitated spermatozoa, the only difference being the level of maximum decondensation achieved. Heparan sulfate and heparin, but not other GAGs, were able to release protamines from sperm chromatin. Conclusions: Heparan sulfate could be functioning as protamine acceptor in vivo during human sperm nuclear decondensation. © The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Descripción: In spite of its massively parallel architecture [1], the human brain is fundamentally limited if required to perform two tasks at the same time [2, 3]. This limitation can be studied with the psychological refractory period (PRP) paradigm, where two stimuli that require speeded responses occur in close succession [4]. Interference generally takes the form of a delay in the time to respond to the second stimulus [5]. Previous studies suggested that sensory decisions require the accumulation of sensory evidence [6, 7] and that the PRP reflects the inability to form more than one decision at a time [4, 8]. In the present study, we used a psychophysical reverse-correlation technique [9, 10] to measure the time-course of evidence accumulation during the PRP. We found that the accumulation of evidence could occur during the PRP albeit with a reduced efficiency, which implies that multiple decision processes can occur in parallel in the human brain. In addition to the reduced efficiency of evidence accumulation, our results uncover an additional delay in the routing of the decision to motor structures during the PRP, which implies that the process of sensory decision making is separable from the preparation of a motor response [11-13]. © 2012 Elsevier Ltd. All rights reserved.

Descripción: Nasal secretions were studied in 80 allergic adults patients: 16 with intermittent rhinitis and 64 with persistent rhinitis. The percentage of supranuclear stria of ciliated cells with regard to leucocytes was studied by nasal scraping. Four groups of patients were classified according to nasal leucocytic predominance: patients with eosinophilic predominance with eosinophils > 10% in Group A (N=23), patients with abundant neutrophils and eosinophils > 10% in Group B (N=15), patients with scant leucocytes in Group C (N=29), patients with neutrophilic predominance without eosinophils in Group D (N=13). An increase of supranuclear stria percentage was correlated to eosinophils > 10% and also correlated to scant leucocytes. Nevertheless, a significant decrease of supranuclear stria percentage was observed in neutrophilic leukocytosis of bacterial etiology.

Descripción: Human immunodeficiency virus type 1 (HIV-1) Gag selects for and mediates genomic RNA (vRNA) encapsidation into progeny virus particles. The host protein, Staufen1 interacts directly with Gag and is found in ribonucleoprotein (RNP) complexes containing vRNA, which provides evidence that Staufen1 plays a role in vRNA selection and encapsidation. In this work, we show that Staufen1, vRNA and Gag are found in the same RNP complex. These cellular and viral factors also colocalize in cells and constitute novel Staufen1 RNPs (SHRNPs) whose assembly is strictly dependent on HIV-1 expression. SHRNPs are distinct from stress granules and processing bodies, are preferentially formed during oxidative stress and are found to be in equilibrium with translating polysomes. Moreover, SHRNPs are stable, and the association between Staufen1 and vRNA was found to be evident in these and other types of RNPs. We demonstrate that following Staufen1 depletion, apparent supraphysiologic-sized SHRNP foci are formed in the cytoplasm and in which Gag, vRNA and the residual Staufen1 accumulate. The depletion of Staufen1 resulted in reduced Gag levels and deregulated the assembly of newly synthesized virions, which were found to contain several-fold increases in vRNA, Staufen1 and other cellular proteins. This work provides new evidence that Staufen1-containing HIV-1 RNPs preferentially form over other cellular silencing foci and are involved in assembly, localization and encapsidation of vRNA.

Descripción: While there is broad consensus about the structural similarities between language and music, comparably less attention has been devoted to semantic correspondences between these two ubiquitous manifestations of human culture. We have investigated the relations between music and a narrow and bounded domain of semantics: the words and concepts referring to taste sensations. In a recent work, we found that taste words were consistently mapped to musical parameters. Bitter is associated with low-pitched and continuous music (legato), salty is characterized by silences between notes (staccato), sour is high pitched, dissonant and fast and sweet is consonant, slow and soft (Mesz2011). Here we extended these ideas, in a synergistic dialog between music and science, investigating whether music can be algorithmically generated from taste-words. We developed and implemented an algorithm that exploits a large corpus of classic and popular songs. New musical pieces were produced by choosing fragments from the corpus and modifying them to minimize their distance to the region in musical space that characterizes each taste. In order to test the capability of the produced music to elicit significant associations with the different tastes, musical pieces were produced and judged by a group of non musicians. Results showed that participants could decode well above chance the taste-word of the composition. We also discuss how our findings can be expressed in a performance bridging music and cognitive science. © 2012 Mesz, Sigman and Trevisan.

Descripción: Papillomaviruses are small DNA tumor viruses that infect mammalian hosts, with consequences from benign to cancerous lesions. The Early protein 2 is the master regulator for the virus life cycle, participating in gene transcription, DNA replication, and viral episome migration. All of these functions rely on primary target recognition by its dimeric DNA-binding domain. In this work, we performed molecular dynamics simulations in order to gain insights into the structural dynamics of the DNA-binding domains of two prototypic strains, human papillomavirus strain 16 and the bovine papillomavirus strain 1. The simulations underline different dynamic features in the two proteins. The human papillomavirus strain 16 domain displays a higher flexibility of the β2-β3 connecting loop in comparison with the bovine papillomavirus strain 1 domain, with a consequent effect on the DNA-binding helices, and thus on the modulation of DNA recognition. A compact β-barrel is found in human papillomavirus strain 16, whereas the bovine papillomavirus strain 1 protein is characterized by a loose β-barrel with a large number of cavities filled by water, which provides great flexibility. The rigidity of the human papillomavirus strain 16 β-barrel prevents protein deformation, and, as a consequence, deformable spacers are the preferred targets in complex formation. In contrast, in bovine papillomavirus strain 1, a more deformable β-barrel confers greater adaptability to the protein, allowing the binding of less flexible DNA regions. The flexibility data are confirmed by the experimental NMR S2 values, which are reproduced well by calculation. This feature may provide the protein with an ability to discriminate between spacer sequences. Clearly, the deformability required for the formation of the Early protein 2 C-terminal DNA-binding domain-DNA complexes of various types is based not only on the rigidity of the base sequences in the DNA spacers, but also on the intrinsic deformability properties of each domain. © 2007 The Authors.

Descripción: Background: Human papillomavirus (HPV) is the main causative agent of cervical cancer, particularly high risk strains such us HPV-16, -18 and -31. The viral encoded E2 protein acts as a transcriptional modulator and exerts a key role in viral DNA replication. Thus, E2 constitutes an attractive target for developing antiviral agents. E2 is a homodimeric protein that interacts with the DNA target through an α-helix of each monomer. However, a peptide corresponding to the DNA recognition helix of HPV-16 E2 binds DNA with lower affinity than its full-length DNA binding domain. Therefore, in an attempt to promote the DNA binding of the isolated peptide, we have designed a conjugate compound of the E2 α-helix peptide and a derivative of the antibiotic distamycin, which involves simultaneous minor- and major-groove interactions. Methodology/Principal Findings: An E2 α-helix peptide-distamycin conjugate was designed and synthesized. It was characterized by NMR and CD spectroscopy, and its DNA binding properties were investigated by CD, DNA melting and gel shift experiments. The coupling of E2 peptide with distamycin does not affect its structural properties. The conjugate improves significantly the affinity of the peptide for specific DNA. In addition, stoichiometric amounts of specific DNA increase meaningfully the helical population of the peptide. The conjugate enhances the DNA binding constant 50-fold, maintaining its specificity. Conclusions/Significance: These results demonstrate that peptide-distamycin conjugates are a promising tool to obtain compounds that bind the E2 target DNA-sequences with remarkable affinity and suggest that a bipartite major/minor groove binding scaffold can be a useful approach for therapeutic treatment of HPV infection. © 2011 Wetzler et al.

Descripción: As for any solid tumour, pituitary adenoma expansion is dependent on neovascularization through angiogenesis. In this process, vascular endothelial growth factor (VEGF) and its receptors VEGFR-1, VEGFR-2 and neuropilin-1 (NRP-1) may play an outstanding role. The intention of this work was to study the expression/localization and possible function of VEGF receptors in pituitary adenomas. VEGF receptor mRNA and protein expression was studied by in situ hybridization, immunohistochemistry and RT-PCR in 6 normal human pituitaries, 39 human pituitary adenomas and 4 rodent pituitary adenoma cell lines. VEGFR-1 expressing somatotroph MtT-S cells were used as a model to study the role of VEGF on cell proliferation and to elucidate the underlying mechanism of action. In normal pituitaries, VEGFR-1 was detected in endocrine cells, whereas VEGFR-2 and NRP-1 were exclusively expressed in endothelial cells. In pituitary tumours, a heterogeneous VEGFR expression pattern was observed by IHC. VEGFR-1, VEGFR-2 and NRP-1 were detected in 24, 18 and 17 adenomas respectively. In the adenomas, VEGFR-1 was expressed in epithelial tumour cells and VEGFR-2/NRP-1 in vessel endothelial cells. Functional studies in VEGFR-1-positive MtT-S cells showed that the ligands of VIEGFR-1 significantly stimulated cell proliferation. This effect was mediated through the phosphatidylinositol-3-kinase-signalling pathway and involves induction of cyclin D1 and Bcl-2. Based on our results, we speculate that the ligands of VEGF receptors, such as VEGF-A and placenta growth factor, not only play a role in angiogenesis in pituitary adenomas, but also affect the growth of pituitary tumour cells through VEGFR-1. © 2006 Society for Endocrinology.

Descripción: The Antarctic icefish Chaenocephalus aceratus lacks the globins common to most vertebrates, hemoglobin and myoglobin, but has retained neuroglobin in the brain. This conserved globin has been cloned, over-expressed and purified. To highlight similarities and differences, the structural features of the neuroglobin of this colourless-blooded fish were compared with those of the well characterised human neuroglobin as well as with the neuroglobin from the retina of the red blooded, hemoglobin and myoglobin-containing, closely related Antarctic notothenioid Dissostichus mawsoni. A detailed structural and functional analysis of the two Antarctic fish neuroglobins was carried out by UV-visible and Resonance Raman spectroscopies, molecular dynamics simulations and laser-flash photolysis. Similar to the human protein, Antarctic fish neuroglobins can reversibly bind oxygen and CO in the Fe 2+ form, and show six-coordination by distal His in the absence of exogenous ligands. A very large and structured internal cavity, with discrete docking sites, was identified in the modelled three-dimensional structures of the Antarctic neuroglobins. Estimate of the free-energy barriers from laser-flash photolysis and Implicit Ligand Sampling showed that the cavities are accessible from the solvent in both proteins. Comparison of structural and functional properties suggests that the two Antarctic fish neuroglobins most likely preserved and possibly improved the function recently proposed for human neuroglobin in ligand multichemistry. Despite subtle differences, the adaptation of Antarctic fish neuroglobins does not seem to parallel the dramatic adaptation of the oxygen carrying globins, hemoglobin and myoglobin, in the same organisms. © 2012 Giordano et al.

Descripción: By taking advantage of the extreme stability of a protein-DNA complex, we have obtained two highly specific monoclonal antibodies against a predetermined palindromic DNA sequence corresponding to the binding site of the E2 transcriptional regulator of the human papillomavirus (HPV-16). The purified univalent antibody fragments bind to a double-stranded DNA oligonucleotide corresponding to the E2 binding site in solution with dissociation constants in the low and subnanomolar range. This affinity matches that of the natural DNA binding domain and is severalfold higher than the affinity of a homologous bovine E2 C-terminal domain (BPV-1) for the same DNA. These antibodies discriminate effectively among a number of double- and single-stranded synthetic DNAs with factors ranging from 125-to 20,000-fold the dissociation constant of the specific DNA sequence used in the immunogenic protein-DNA complex. Moreover, they are capable of fine specificity tuning, since they both bind less tightly to another HPV-16 E2 binding site, differing in only 1 base pair in a noncontact flexible region. Beyond the relevance of obtaining a specific anti-DNA response, these results provide a first glance at how DNA as an antigen is recognized specifically by an antibody. The accuracy of the spectroscopic method used for the binding analysis suggests that a detailed mechanistic analysis is attainable.

Descripción: Background: Self-assembly is a common theme in proteins of unrelated sequences or functions. The human papillomavirus E7 oncoprotein is an extended dimer with an intrinsically disordered domain, that can form large spherical oligomers. These are the major species in the cytosol of HPV transformed and cancerous cells. E7 binds to a large number of targets, some of which lead to cell transformation. Thus, the assembly process not only is of biological relevance, but represents a model system to investigate a widely distributed mechanism. Methodology/Principal Findings: Using various techniques, we monitored changes in secondary, tertiary and quaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formation of a monomeric "Z-nucleus" after zinc removal, followed by an elongation phase consisting of sequential second-order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 μM protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a "conformation editing" mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5 kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsically disordered domain stabilizes the oligomer, preventing an insoluble amyloid route. Conclusion: We described a controlled ordered mechanism with features in common with soluble amyloid precursors, chaperones, and other spherical oligomers, thus sharing determining factors for symmetry, size and shape. In addition, such a controlled and discrete polymerization reaction provides a valuable tool for nanotechnological applications. Finally, its increased immunogenicity related to its supramolecular structure is the basis for the development of a promising therapeutic vaccine candidate for treating HPV cancerous lesions. © 2012 Smal et al.

Descripción: Kaposi's sarcoma (KS), a multifocal vascular neoplasm linked to human herpesvirus-8 (HHV-8/KS-associated herpesvirus [KSHV]) infection, is the most common AIDS-associated malignancy. Clinical management of KS has proven to be challenging because of its prevalence in immunosuppressed patients and its unique vascular and inflammatory nature that is sustained by viral and host-derived paracrine-acting factors primarily released under hypoxic conditions. We show that interactions between the regulatory lectin galectin-1 (Gal-1) and specific target N-glycans link tumor hypoxia to neovascularization as part of the pathogenesis of KS. Expression of Gal-1 is found to be a hallmark of human KS but not other vascular pathologies and is directly induced by both KSHV and hypoxia. Interestingly, hypoxia induced Gal-1 through mechanisms that are independent of hypoxia-inducible factor (HIF) 1α and HIF-2α but involved reactive oxygen species-dependent activation of the transcription factor nuclear factor κB. Targeted disruption of Gal-1-N-glycan interactions eliminated hypoxia-driven angiogenesis and suppressed tumorigenesis in vivo. Therapeutic administration of a Gal-1-specific neutralizing mAb attenuated abnormal angiogenesis and promoted tumor regression in mice bearing established KS tumors. Given the active search for HIF-independent mechanisms that serve to couple tumor hypoxia to pathological angiogenesis, our findings provide novel opportunities not only for treating KS patients but also for understanding and managing a variety of solid tumors. © 2012 Croci et al.