Use of top-down and bottom-up fourier transform ion cyclotron resonance mass spectrometry for mapping calmodulin sites modified by platinum anticancer drugs

Calmodulin (CaM) is a highly conserved, ubiquitous, calcium-binding protein; it binds to and regulates many different protein targets, thereby functioning as a calcium sensor and signal transducer. CaM contains 9 methionine (Met), 1 histidine (His), 17 aspartic acid (Asp), and 23 glutamine acid (Glu) residues, all of which can potentially react with platinum compounds; thus, one-third of the CaM sequence is a possible binding target of platinum anticancer drugs, which represents a major challenge for identification of specific platinum modification sites. Here, top-down electron capture dissociation (ECD) was used to elucidate the transition metal–platinum(II) modification sites. By using a combination of top-down and bottom-up mass spectrometric (MS) approaches, 10 specific binding sites for mononuclear complexes, cisplatin and [Pt(dien)Cl]Cl, and dinuclear complex [{cis-PtCl2(NH3)}2(μ-NH2(CH2)4NH2)] on CaM were identified. High resolution MS of cisplatin-modified CaM revealed that cisplatin mainly targets Met residues in solution at low molar ratios of cisplatin–CaM (2:1), by cross-linking Met residues. At a high molar ratio of cisplatin:CaM (8:1), up to 10 platinum(II) bind to Met, Asp, and Glu residues. [{cis-PtCl2(NH3)}2(μ-NH2(CH2)4NH2)] forms mononuclear adducts with CaM. The alkanediamine linker between the two platinum centers dissociates due to a trans-labilization effect. [Pt(dien)Cl]Cl forms {Pt(dien)}2+ adducts with CaM, and the preferential binding sites were identified as Met51, Met71, Met72, His107, Met109, Met124, Met144, Met145, Glu45 or Glu47, and Asp122 or Glu123. The binding of these complexes to CaM, particularly when binding involves loss of all four original ligands, is largely irreversible which could result in their failure to reach the target DNA or be responsible for unwanted side-effects during chemotherapy. Additionally, the cross-linking of cisplatin to CaM might lead to the loss of the biological function of CaM or CaM–Ca2+ due to limiting the flexibility of the CaM or CaM–Ca2+ complex to recognize target proteins or blocking the binding region of target proteins to CaM

Efecto del peso del bulbo sobre la floracion y estudios de diferenciacion floral de Herbertia lahue (MOL.) Goldbl.

Resumen (Spanish, English)61 p.Debido al potencial ornamental que presenta la especie nativa chilena Herbertia lahue (Mol.) Goldbl., se realizaron estudios tendientes a dilucidar el efecto del peso de su bulbo sobre aspectos florales, fenológicos y multiplicativos de las plantas, efectuándose además un experimento complementario con el objetivo de determinar la influencia de la remoción de la túnica del bulbo sobre el comportamiento de las plantas. Ambos experimentos fueron conducidos bajo un diseño completamente al azar de un factor (con cinco categorías de peso 0,15 ± 0,1 g; 0,35 ± 0,1 g; 0,75 ± 0,1 g; 1,35 ± 0,1 g y 1,75 ± 0,1 g) y dos factores (con dos categorías de peso (0,85 ± 0,15 g y 1,35 ± 0,15 g, con y sin túnica), respectivamente. Paralelo a éstos se realizó mediante microscopía electrónica de barrido, el estudio de su diferenciación floral, asociando la formación de la yema floral al estado de desarrollo de la planta y los días transcurridos desde plantación. Los experimentos se realizaron en uno de los invernaderos fríos de polietileno de la Universidad de Talca campus Lircay, durante el periodo febrero de 2000 a enero de 2001. Se determinó que el máximo porcentaje de floración (97%-100%) se logró con bulbos de peso igual o superior a 0,75 ± 0,1 g; bulbos de 1,75 ± 0,1 g proporcionaron la mayor cantidad promedio de flores, cercana a tres; el mayor diámetro floral y longitud del tallo floral se obtuvo con bulbos de peso igual o superior a 1,35 ± 0,1 g; mientras que el diámetro del tallo no fue influenciado por el peso del bulbo. Aspectos fenológicos tales como los días transcurridos desde plantación a emergencia y a senescencia no fueron influenciados por el peso del bulbo, a diferencia de los días transcurridos desde plantación a floración, donde bulbos de peso igual o superior a 0,75 ± 0,1 g adelantaron su floración en comparación al menor peso de bulbo floral, transcurriendo en promedio 258 días desde plantación. El periodo de floración, es decir los días transcurridos desde la antesis de la primera flor hasta la antesis de la última, alcanzó un máximo de 22 días en bulbos de 1,75 ± 0,1 g. Bulbos de 0,15 ± 0,1 g presentaron 9,6 hojas emitidas, mientras que bulbos de mayor peso presentaron mas de 12 hojas, valor significativamente superior. El peso final de los bulbos cosechados fue superior en bulbos de mayor peso inicial, sin embargo se observó una ganancia mayor de peso, en bulbos de menor peso inicial, a diferencia de bulbos de peso igual o mayor a 1,35 ± 0,1 g los que presentaron pérdidas de peso. La capacidad multiplicativa de Herbertia lahue no se vio influenciada por el peso de los bulbos, alcanzando la tasa de multiplicación un valor levemente superior a 1,0 sólo en bulbos de peso igual o mayor a 1,35 ± 0,1 g. El comportamiento de las plantas no se vio afectado por la remoción de la túnica del bulbo al momento de la plantación, presentando la planta características florales, fenológicas y multiplicativas similares en bulbos con presencia y ausencia de túnica. La iniciación y diferenciación floral de Herbertia lahue tuvo lugar en el mes de septiembre, alrededor de 27 semanas luego de la plantación del bulbo, presentando la planta entre 8 a 10 hojas formadas

Agreement on the perception of moral character

This study tested for inter-judge agreement on moral character. A sample of students and community members rated their own moral character using a measure that tapped six moral character traits. Friends, family members, and/or acquaintances rated these targets on the same traits. Self/other and inter-informant agreement was found at the trait level for both a general character factor and for residual variance explained by individual moral character traits, as well as at the individual level (judges agreed on targets’ “moral character profiles”). Observed inter-judge agreement constitutes evidence for the existence of moral character, and raises questions about the nature of moral character traits

Osteoma of external acoustic meato: report of nine cases and literature review

The osteoma of external acoustic meatus corresponds to an abnormal and benign bony growth, that is developed under the skin of the auditory canal and that can cause total or partial obstruction of the external acoustic meatus. STUDY DESIGN: Serie study. MATERIAL AND METHOD: We reported in this study nine cases of osteoma of external acoustic meatus. The patients' ages varied between 23 and 70 years old, being the average age 40,6 years. These patients were 5 of the feminine sex and 4 were of the masculine sex. RESULTS: The more common symptoms were hipoacusia, caused by the partial or total obstruction of the canal, appealing external otitis and also the accumulation of cerumen deep in the external auditory canal with difficult removal. CONCLUSION: We recommended in the presented cases surgical treatment, which had good evolution, with no recidivism.Osteoma de meato acústico externo corresponde a um crescimento ósseo anormal e benigno, que se desenvolve abaixo da pele do conduto e pode causar obstrução total ou parcial do meato acústico externo. FORMA DE ESTUDO: Estudo de série. MATERIAL E MÉTODO: Apresentamos neste estudo nove casos de osteoma de meato acústico externo. A idade dos pacientes variou de 23 anos (idade mínima) e 70 anos (idade máxima), sendo a média 40,6 anos. Desses nove pacientes, 5 eram do sexo feminino e 4 eram do sexo masculino. RESULTADO: Os sintomas mais comuns foram: hipoacusia, causada pela obstrução parcial ou total do conduto, otites externas recorrentes e também impactação de cerume de difícil remoção. CONCLUSÃO: A conduta nos casos apresentados foi cirúrgica, com boa evolução, não ocorrendo recidivas.UNIFESP-EPMHospital Paulista de OtorrinolaringologiaUNIFESP, EPMSciEL

Does native Trypanosoma cruzi calreticulin mediate growth inhibition of a mammary tumor during infection?

Indexación: Web of Science.Background: For several decades now an antagonism between Trypanosoma cruzi infection and tumor development has been detected. The molecular basis of this phenomenon remained basically unknown until our proposal that T. cruzi Calreticulin (TcCRT), an endoplasmic reticulum-resident chaperone, translocated-externalized by the parasite, may mediate at least an important part of this effect. Thus, recombinant TcCRT (rTcCRT) has important in vivo antiangiogenic and antitumor activities. However, the relevant question whether the in vivo antitumor effect of T. cruzi infection is indeed mediated by the native chaperone (nTcCRT), remains open. Herein, by using specific modified anti-rTcCRT antibodies (Abs), we have neutralized the antitumor activity of T. cruzi infection and extracts thereof, thus identifying nTcCRT as a valid mediator of this effect. Methods: Polyclonal anti-rTcCRT F(ab')(2) Ab fragments were used to reverse the capacity of rTcCRT to inhibit EAhy926 endothelial cell (EC) proliferation, as detected by BrdU uptake. Using these F(ab')(2) fragments, we also challenged the capacity of nTcCRT, during T. cruzi infection, to inhibit the growth of an aggressive mammary adenocarcinoma cell line (TA3-MTXR) in mice. Moreover, we determined the capacity of anti-rTcCRT Abs to reverse the antitumor effect of an epimastigote extract (EE). Finally, the effects of these treatments on tumor histology were evaluated. Results: The rTcCRT capacity to inhibit ECs proliferation was reversed by anti-rTcCRT F(ab')(2) Ab fragments, thus defining them as valid probes to interfere in vivo with this important TcCRT function. Consequently, during infection, these Ab fragments also reversed the in vivo experimental mammary tumor growth. Moreover, anti-rTcCRT Abs also neutralized the antitumor effect of an EE, again identifying the chaperone protein as an important mediator of this anti mammary tumor effect. Finally, as determined by conventional histological parameters, in infected animals and in those treated with EE, less invasive tumors were observed while, as expected, treatment with F(ab')(2) Ab fragments increased malignancy. Conclusion: We have identified translocated/externalized nTcCRT as responsible for at least an important part of the anti mammary tumor effect of the chaperone observed during experimental infections with T. cruzi.http://bmccancer.biomedcentral.com/articles/10.1186/s12885-016-2764-

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that processFil: Salinas Ojeda, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ortiz Flores, Rodolfo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Distel, Jesús Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Aguilera, Milton Osmar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Beron, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Somatostatin subtype-2 receptor-targeted metal-based anticancer complexes

Conjugates of a dicarba analogue of octreotide, a potent somatostatin agonist whose receptors are overexpressed on tumor cells, with [PtCl 2(dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (3), [(η 6-bip)Os(4-CO 2-pico)Cl] (bip = biphenyl, pico = picolinate) (4), [(η 6-p-cym)RuCl(dap)] + (p-cym = p-cymene) (5), and [(η 6-p-cym)RuCl(imidazole-CO 2H)(PPh 3)] + (6), were synthesized by using a solid-phase approach. Conjugates 3-5 readily underwent hydrolysis and DNA binding, whereas conjugate 6 was inert to ligand substitution. NMR spectroscopy and molecular dynamics calculations showed that conjugate formation does not perturb the overall peptide structure. Only 6 exhibited antiproliferative activity in human tumor cells (IC 50 = 63 ± 2 μ in MCF-7 cells and IC 50 = 26 ± 3 μ in DU-145 cells) with active participation of somatostatin receptors in cellular uptake. Similar cytotoxic activity was found in a normal cell line (IC 50 = 45 ± 2.6 μ in CHO cells), which can be attributed to a similar level of expression of somatostatin subtype-2 receptor. These studies provide new insights into the effect of receptor-binding peptide conjugation on the activity of metal-based anticancer drugs, and demonstrate the potential of such hybrid compounds to target tumor cells specifically. © 2012 American Chemical Society

PCR-mediated detection of acidophilic, bioleaching-associated bacteria

The detection of acidophilic microorganisms from mining environments by culture methods is time consuming and unreliable. Several PCR approaches were developed to amplify small-subunit rRNA sequences from the DNA of six bacterial phylotypes associated with acidic mining environments, permitting the detection of the target DNA at concentrations as low as 10 fg

Towards generalized measures grasping CA dynamics

In this paper we conceive Lyapunov exponents, measuring the rate of separation between two initially close configurations, and Jacobians, expressing the sensitivity of a CA's transition function to its inputs, for cellular automata (CA) based upon irregular tessellations of the n-dimensional Euclidean space. Further, we establish a relationship between both that enables us to derive a mean-field approximation of the upper bound of an irregular CA's maximum Lyapunov exponent. The soundness and usability of these measures is illustrated for a family of 2-state irregular totalistic CA

Talk the talk, walk the walk: Defining Critical Race Theory in research

Over the last decade there has been a noticeable growth in published works citing Critical Race Theory (CRT). This has led to a growth in interest in the UK of practical research projects utilising CRT as their framework. It is clear that research on 'race' is an emerging topic of study. What is less visible is a debate on how CRT is positioned in relation to methodic practice, substantive theory and epistemological underpinnings. The efficacy of categories of data gathering tools, both traditional and non-traditional is a discussion point here to explore the complexities underpinning decisions to advocate a CRT framework. Notwithstanding intersectional issues, a CRT methodology is recognisable by how philosophical, political and ethical questions are established and maintained in relation to racialised problematics. This paper examines these tensions in establishing CRT methodologies and explores some of the essential criteria for researchers to consider in utilising a CRT framework. © 2012 Copyright Taylor and Francis Group, LLC