MPK-09, a small molecule inspired from bioactive styryllactone restores the wild-type function of mutant p53

In the search for more efficacious and less toxic cancer drugs, the tumor suppressor p53 protein has long been a desirable therapeutic target. In the recent past, few independent studies have demonstrated that the antitumor activity of wild-type p53 can be restored in cancer cells harboring mutant form of p53 using small molecule activators. In this study, we describe a novel small molecule MPK-09, which is selective and highly potent against allele specific p53 mutations mainly, R175H, R249S, R273H, R273C, and E285K. Except E285K, all other mutations tested are among the six ``hot spot'' p53 mutations reported in majority of human cancer. Furthermore, our study conclusively demonstrates that the apoptotic activity of the small molecule MPK-09 against cancer cells harboring R273C and E285K mutations is due to restoration of the wild-type conformation to the corresponding mutant form of p53.

Biodegradation of sodium isopropyl xanthate by Paenibacillus polymyxa and Pseudomonas putida

Biodegradation of sodium isopropyl xanthate using two types of bacterial strains, Paenibacillus polymyxa and Pseudomonas putida, is demonstrated. At concentrations higher than 50 mg/L, the presence of xanthate in the growth medium resulted in bacterial toxicity, retarding growth kinetics. Adaptation through serial subculturing in the presence of higher xanthate concentrations resulted in the development of xanthate-tolerant bacterial strains. Stress proteins secreted by bacterial cells grown in the presence of xanthate were isolated. Bacterial cells could utilize xanthate as a growth substrate, degrading xanthate species in the process. Acidic metabolic products generated by bacterial metabolism promoted efficient xanthate decomposition. Probable mechanisms for the biodegradation of isopropyl xanthate are illustrated.

A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library

With the development of deep sequencing methodologies, it has become important to construct site saturation mutant (SSM) libraries in which every nucleotide/codon in a gene is individually randomized. We describe methodologies for the rapid, efficient, and economical construction of such libraries using inverse polymerase chain reaction (PCR). We show that if the degenerate codon is in the middle of the mutagenic primer, there is an inherent PCR bias due to the thermodynamic mismatch penalty, which decreases the proportion of unique mutants. Introducing a nucleotide bias in the primer can alleviate the problem. Alternatively, if the degenerate codon is placed at the 5' end, there is no PCR bias, which results in a higher proportion of unique mutants. This also facilitates detection of deletion mutants resulting from errors during primer synthesis. This method can be used to rapidly generate SSM libraries for any gene or nucleotide sequence, which can subsequently be screened and analyzed by deep sequencing. (C) 2013 Elsevier Inc. All rights reserved.

Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

Huntington's disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK. (C) 2014 Elsevier Inc. All rights reserved.

Curcumin Pyrazole and its derivative (N-(3-Nitrophenylpyrazole) Curcumin inhibit aggregation, disrupt fibrils and modulate toxicity of Wild type and Mutant alpha-Synuclein

Accumulating evidence suggests that deposition of neurotoxic a-synuclein aggregates in the brain during the development of neurodegenerative diseases like Parkinson's disease can be curbed by anti-aggregation strategies that either disrupt or eliminate toxic aggregates. Curcumin, a dietary polyphenol exhibits anti-amyloid activity but the use of this polyphenol is limited owing to its instability. As chemical modifications in curcumin confiscate this limitation, such efforts are intensively performed to discover molecules with similar but enhanced stability and superior properties. This study focuses on the inhibitory effect of two stable analogs of curcumin viz. curcumin pyrazole and curcumin isoxazole and their derivatives against a-synuclein aggregation, fibrillization and toxicity. Employing biochemical, biophysical and cell based assays we discovered that curcumin pyrazole (3) and its derivative N-(3-Nitrophenylpyrazole) curcumin (15) exhibit remarkable potency in not only arresting fibrillization and disrupting preformed fibrils but also preventing formation of A11 conformation in the protein that imparts toxic effects. Compounds 3 and 15 also decreased neurotoxicity associated with fast aggregating A53T mutant form of a-synuclein. These two analogues of curcumin described here may therefore be useful therapeutic inhibitors for the treatment of a-synuclein amyloidosis and toxicity in Parkinson's disease and other synucleinopathies.

A novel Monoclonal Antibody against Notch1 Targets Leukemia-associated Mutant Notch1 and Depletes Therapy Resistant Cancer Stem Cells in Solid Tumors

Higher Notch signaling is known to be associated with hematological and solid cancers. We developed a potential immunotherapeutic monoclonal antibody (MAb) specific for the Negative Regulatory Region of Notch1 (NRR). The MAb604.107 exhibited higher affinity for the ``Gain-offunction'' mutants of Notch1 NRR associated with T Acute lymphoblastic Leukemia (T-ALL). Modeling of the mutant NRR with 12 amino-acid insertion demonstrated ``opening'' resulting in exposure of the S2-cleavage site leading to activated Notch1 signaling. The MAb, at low concentrations (1-2 mu g/ml), inhibited elevated ligand-independent Notch1 signaling of NRR mutants, augmented effect of Thapsigargin, an inhibitor of mutant Notch1, but had no effect on the wild-type Notch1. The antibody decreased proliferation of the primary T-ALL cells and depleted leukemia initiating CD34/CD44 high population. At relatively high concentrations, (10-20 mu g/ml), the MAb affected Notch1 signaling in the breast and colon cancer cell lines. The Notch-high cells sorted from solid-tumor cell lines exhibited characteristics of cancer stem cells, which were inhibited by the MAb. The antibody also increased the sensitivity to Doxorubucinirubicin. Further, the MAb impeded the growth of xenografts from breast and colon cancer cells potentiated regression of the tumors along with Doxorubucin. Thus, this antibody is potential immunotherapeutic tool for different cancers.

Evaluación de enmiendas y microorganismos antagonistas para el manejo de pseudomonas solanacearum E.F. Smith en cultivo de tomate (Lycopersicon esculentum L.)

Se estudió el efecto de dos enmiendas y tres microorganismos como posibles factores supresívos y antagonistas respectivamente al agente causal de la marchitez bacteriana Pseudomonas solanacearum E.F.Srnlth. Los experimentos fueron conducidos en campo e invernadero en el Centro Agronómíco Tropical de Investigación y Enseñanza (CATIE), Turrialba, Costa Rica. Se establecieron en diseño de parcelas divididas con cuatro repeticiones en el campo y tres en el invernadero. Las parcelas principales se dispusieron en bloques completamente al azar. Se evaluaron tres tratamientos (compost comercial, cal dolomítica y un tratamiento sin enmiendas) en las parcelas principales y ocho tratamientos (Pseudomonas cepacia, Bacillus cereus y Glomus occultum solos y en combínación) en las subparcelas. Los tratamientos con enmiendas se asignaron a las parcelas principales, mientras que los tratamientos con antagonistas se asignaron a las subparcelas. Las variables evaluadas fueron incidencia y severidad de la enfermedad y área bajo la curva de progreso de la enfermedad (ABCPE). Se determinó el efecto de las enmiendas sobre las poblaciones de P. solanacearum en tres momentos. En el campo no hubo diferencias significativas en las varia~ bles evaluadas. Las poblaciones de P. solanocearum fueron reducidas significativamente en el suelo. En invernadero, las enmiendas redujeron significativamente la severidad de la enfermedad y el ABCPE. Los antagonistas no redujeron significativamente la incidencia y la severidad en el campo e invernadero.

Estudio de tres métodos para el diagnóstico de Pseudomonas solanacearum E. Smith en suelos cultivados con papa (Solanum tuberosum L.) en la I y VI regiones de Nicaragua

Para evaluar la eficiencia del método historial de campo el diagnóstico del grado de infección de suelos por Pseudimonas solanacearum se recolectaron 20 muestras de suelo en las regiones I y VI en Nicaragua. De cada región en base al historial de campo se colectaron tres hasta cuatro muestras de suelos con grados alto, medio y cero grado de infección por PS. Solanacearum respectivamente. La recolección de las muestras de suelos se realizaron en los meses de Mayo y en Octubre de 1989 efectuándose una recolección en época seca (Mayo) y otra época lluviosa (Octubre) sobre los mismos suelos. A los suelos colectados en época seca se les realizó conteo de colonias de PS. Solanacearum por método In vitro y con los colectados en época lluviosa además del conteo In Vitro se realizaron bioensayos con plantas de tomate, tabaco y berengena. Los resultados del análisis In Vitro sobre las muestras de suelos en platos Petri indicaron que todas las muestras de suelos colectados presentaron algún número de colonias de PS. Solanacearum por lo cual no existió ningún suelo libre de infestación según el historial de campo, 19 de los 20 suelos colectados en época lluviosa posibilitaron el desarrollo de síntomas de la enfermedad, resultando las plantas de berengena las más susceptibles en menor tiempo por el método del bioensayo. En suelos colectados en época seca el número de colonias fue mayor en relación al número de colonias encontradas en época lluviosa en los mismos suelos. Aunque no fue posible encontrar una correlación efectivo entre el diagnóstico del grado de infestación del suelo por el método del historial de . campo y los otros dos métodos de estudio, se observó algún grado de asociación entre el diagnóstico por el historial y conteo In Vitro para el caso de los suelos de la primera región cuando se trató de suelos catalogados como alto y medio grados de infestación, según el método historial ele campo únicamente.

Structure-function studies of serine hydrolases: synthesis of a gene for ∝-lytic protease and the purification and characterization of a mutant β-lactamase

The author has constructed a synthetic gene for ∝-lytic protease. Since the DNA sequence of the protein is not known, the gene was designed by using the reverse translation of ∝-lytic protease's amino acid sequence. Unique restriction sites are carefully sought in the degenerate DNA sequence to aid in future mutagenesis studies. The unique restriction sites are designed approximately 50 base pairs apart and their appropriate codons used in the DNA sequence. The codons used to construct the DNA sequence of ∝-lytic protease are preferred codons in E-coli or used in the production of β-lactamase. Codon usage is also distributed evenly to ensure that one particular codon is not heavily used. The gene is essentially constructed from the outside in. The gene is built in a stepwise fashion using plasmids as the vehicles for the ∝-lytic oligomers. The use of plasmids allows the replication and isolation of large quantities of the intermediates during gene synthesis. The ∝-lytic DNA is a double-stranded oligomer that has sufficient overhang and sticky ends to anneal correctly in the vector. After six steps of incorporating ∝-lytic DNA, the gene is completed and sequenced to ensure that the correct DNA sequence is present and that no mutations occurred in the structural gene.

β-lactamase is the other serine hydrolase studied in this thesis. The author used the class A RTEM-1 β- lactamase encoded on the plasmid pBR322 to investigate the roll of the conserved threonine residue at position 71. Cassette mutagenesis was previously used to generate all possible amino acid substitutions at position 71. The work presented here describes the purification and kinetic characterization of a T71H mutant previously constructed by S. Schultz. The mutated gene was transferred into plasmid pJN for expression and induced with IPTG. The enzyme is purified by column chromatography and FPLC to homogeneity. Kinetic studies reveal that the mutant has lower k_(cat) values on benzylpenicillin, cephalothin and 6-aminopenicillanic acid but no changes in k_m except for cephalothin which is approximately 4 times higher. The mutant did not change siginificantly in its pH profile compared to the wild-type enzyme. Also, the mutant is more sensitive to thermal denaturation as compared to the wild-type enzyme. However, experimental evidence indicates that the probable generation of a positive charge at position 71 thermally stabilized the mutant.

Avaliação dos efeitos da disponibilidade de luz sobre o crescimento e competição entre cepas de Planktothrix agardhii e Microcystis aeruginosa

As cianobactérias Microcystis aeruginosa e Planktothrix agardhii são espécies formadoras de florações comuns em ecossistemas aquáticos eutrofizados. Nestes ambientes, a disponibilidade de luz é um dos fatores determinantes para o desenvolvimento e estruturação da comunidade fitoplanctônica. O presente estudo teve como objetivo investigar o efeito da luz na fisiologia de cepas de cianobactérias (M. aeruginosa e P. agardhii), avaliando o crescimento, a variabilidade inter e intra-específica e a competição por luz. Para tanto foram realizados cultivos estanques em diferentes intensidades luminosas (10, 40, 60, 100 e 500 mol m-2 s-1) e calculadas as taxas de crescimento e os rendimentos máximos das culturas. O requerimento mínimo de luz de cada cepa foi determinado em experimentos com monoculturas em sistemas de cultivo contínuo (quimiostatos) sob condições de limitação de luz. A competição por luz foi avaliada através de experimentos com biculturas em quimiostatos. Foi observada variabilidade intra e inter-específica das cepas, nas diferentes intensidades luminosas testadas. Em 500 μmol m-2 s-1, as cepas de M. aeruginosa obtiveram maior biomassa do que P. agardhii, corroborando a maior sensibilidade de P. agardhii luz. Embora com rendimento máximo menor, P. agardhii cresceu em intensidades luminosas consideradas elevadas para a espécie, 100 e 500 μmol m-2 s-1. Estes resultados evidenciam a capacidade de P. agardhii ocorrer em ambientes com grandes amplitudes de luminosidade. Na intensidade de 10 μmol m-2 s-1, M. aeruginosa e P. agardhii apresentaram crescimento semelhante, demonstrando a habilidade das duas espécies em crescer com pouca luz. Nas monoculturas em quimiostato, sob condições de limitação de luz, as cepas de M. aeruginosa atingiram maior biomassa durante o equilíbrio (steady-state) do que P. agadhii, refletindo uma capacidade suporte mais elevada, enquanto que os valores de requerimento mínimo de luz foram semelhantes entre as duas espécies. Ao competirem, M. aeruginosa superou P. agardhii imediatamente após o início do experimento. Esse rápido crescimento resultou na dominância de M. aeruginosa em todos os pares de cepas testados e, em dois casos, ocorreu exclusão competitiva de P. agardhii. Quando não ocorreu exclusão, P. agardhii conseguiu manter-se no sistema com uma baixa biomassa (ca.15%). Estes resultados ajudam a entender a co-ocorrência destas espécies no ambiente e a dominância de M. aeruginosa mesmo em condições de baixa luminosidade.

Global transcriptional responses of the toxic cyanobacterium, Microcystis aeruginosa, to nitrogen stress, phosphorus stress, and growth on organic matter

Whole transcriptome shotgun sequencing (RNA-seq) was used to assess the transcriptomic response of the toxic cyanobacterium Microcystis aeruginosa during growth with low levels of dissolved inorganic nitrogen (low N), low levels of dissolved inorganic phosphorus (low P), and in the presence of high levels of high molecular weight dissolved organic matter (HMWDOM). Under low N, one third of the genome was differentially expressed, with significant increases in transcripts observed among genes within the nir operon, urea transport genes (urtBCDE), and amino acid transporters while significant decreases in transcripts were observed in genes related to photosynthesis. There was also a significant decrease in the transcription of the microcystin synthetase gene set under low N and a significant decrease in microcystin content per Microcystis cell demonstrating that N supply influences cellular toxicity. Under low P, 27% of the genome was differentially expressed. The Pho regulon was induced leading to large increases in transcript levels of the alkaline phosphatase phoX, the Pst transport system (pstABC), and the sphX gene, and transcripts of multiple sulfate transporter were also significantly more abundant. While the transcriptional response to growth on HMWDOM was smaller (5–22% of genes differentially expressed), transcripts of multiple genes specifically associated with the transport and degradation of organic compounds were significantly more abundant within HMWDOM treatments and thus may be recruited by Microcystis to utilize these substrates. Collectively, these findings provide a comprehensive understanding of the nutritional physiology of this toxic, bloom-forming cyanobacterium and the role of N in controlling microcystin synthesis.