Crystal structure of the catalytic fragment of murine poly(ADP-ribose) polymerase-2.

Poly(ADP-ribose) polymerase-1 (PARP-1) has become an important pharmacological target in the treatment of cancer due to its cellular role as a 'DNA-strand break sensor', which leads in part to resistance to some existing chemo- and radiological treatments. Inhibitors have now been developed which prevent PARP-1 from synthesizing poly(ADP-ribose) in response to DNA-breaks and potentiate the cytotoxicity of DNA damaging agents. However, with the recent discoveries of PARP-2, which has a similar DNA-damage dependent catalytic activity, and additional members containing the 'PARP catalytic' signature, the isoform selectivity and resultant pharmacological effects of existing inhibitors are brought into question. We present here the crystal structure of the catalytic fragment of murine PARP-2, at 2.8 A resolution, and compare this to the catalytic fragment of PARP-1, with an emphasis on providing a possible framework for rational drug design in order to develop future isoform-specific inhibitors

Large-scale Production And Purification Of Recombinant Protein From An Insect Cell/baculovirus System In Erlenmeyer Flasks: Application To The Chicken Poly(adp-ribose) Polymerase Catalytic Domain

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 × 109 infected cells in three liters of culture.308923928Althaus, F.R., Richter, C., ADP-ribosylation of proteins. Enzymology and biological significance (1987) Molecular Biology, Biochemistry and Biophysics, 37, pp. 1-126Lautier, D., Lagueaux, J., Ménard, L., Poirier, G.C., Molecular and biochemical features of poly(ADP-ribose) metabolism (1993) Molecular and Cellular Biochemistry, 122, pp. 171-193Satoh, M.S., Lindahl, T., Role of poly(ADP-ribose) formation in DNA repair (1992) Nature, 356, pp. 356-358Molinete, M., Vermeulen, W., Bürkle, A., Ménissier-de Murcia, J., Küpper, J., Hoeijmakers, J., De Murcia, G., Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells (1993) EMBO Journal, 5, pp. 2109-2117Masson, M., Rolli, V., Dantzer, F., Trucco, C., Schrieber, V., Fribourg, S., Molinete, M., De Murcia, G., Poly(ADP-ribose) polymerase: Structure-function relationship (1995) Biochimie, 77, pp. 456-461Suto, M.L., Suto, M.S., Inhibitors of poly(ADP-ribose) polymerase (ADPRP): Potential chemotherapeutic agents (1991) Drugs of the Future, 16, pp. 723-739Giner, H., Simonin, F., De Murcia, G., Ménissier-de Murcia, J., Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system (1992) Gene, 114, pp. 279-283Simonin, F., Höfferer, L., Panzeter, P., Muller, S., De Murcia, G., Althaus, F.R., The carboxy-terminal domain of human poly(ADP-ribose) polymerase: Overproduction in Escherichia coli, large scale purification and characterization (1993) Journal of Molecular Biology, 268, pp. 13454-13461Murhammer, D.W., Review and patents literature. The use of insect cell cultures for recombinant protein synthesis: Engineering aspects (1991) Applied Biochemistry and Biotechnology, 31, pp. 283-310Goosen, M.F.A., Large-scale insect cell culture (1992) Current Opinion in Biotechnology, 3, pp. 99-104Kamen, A.A., Tom, R.L., Caron, A.W., Chavarie, C., Massie, B., Archambault, J., Culture of insect cells in a helical ribbon impeller bioreactor (1991) Biotechnology and Bioengineering, 38, pp. 619-628Van Lier, F.L.J., Van Den End, E.J., Gooijer, C.D., Vlak, J.M., Tramper, J., Continuous production of baculovirus in a cascade insect-cell reactor (1990) Applied Microbiology and Biotechnology, 33, pp. 43-47Power, J., Greenfield, P.F., Nielsen, L., Reid, S., Modelling the growth and protein production by insect cells following infection by a recombinant baculovirus in suspension culture (1992) Cytotechnology, 9, pp. 149-155Stavroulakis, D.A., Kalogerakis, N., Behie, L.A., Latrou, K., Kinetic data for the BM-5 insect line in repeated-batch suspension culture (1991) Biotechnology, 38, pp. 116-126Neutra, R., Levi, B.-Z., Shoham, Y., Optimization of protein-production by the baculovirus expression vector system in shake flasks (1992) Applied Microbiology and Biotechnology, 37, pp. 74-78Luckow, V.A., Summers, M.D., Signals important for high level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors (1989) Virology, 170, pp. 31-39O'Reily, D.R., Miller, L.K., Luckow, V.A., (1992) Baculovirus Expression System: A Laboratory Manual, , Freeman, New YorkScott, R.L., Blanchard, J.H., Fergusson, C.H.R., Effects of oxygen on recombinant protein production by suspension cultures of Spodoptera frugiperda Sf9 insect cell (1992) Enzyme and Microbial Technology, 14, pp. 798-80

Poly(ADP-ribose) polymerase-1 is a survival factor for radiation-exposed intestinal epithelial stem cells in vivo

Poly(ADP-ribose) polymerase-1 (PARP-1) is a key enzyme mediating the cellular response to DNA strand breaks. It plays a critical role in genomic stability and survival of proliferating cells in culture undergoing DNA damage. Intestinal epithelium is the most proliferative tissue in the mammalian body and its stem cells show extreme sensitivity to low-level genotoxic stress. We investigated the role of PARP-1 in the in vivo damage response of intestinal stem cells in crypts of PARP-1-/- and control mice following whole-body γ-irradiation (1 Gy). In the PARP-1-/- mice there was a significant delay during the first 6 h in the transient p53 accumulation in stem cells whereas an increased number of cells were positive for p21CIP1/WAF1. Either no or only marginal differences were noted in MDM2 expression, apoptosis, induction of or recovery from mitotic blockage, or inhibition of DNA synthesis. We further observed a dose-dependent reduction in crypt survival measured at 4 days post-irradiation in control mice, and this crypt-killing effect was significantly potentiated in PARP-1-/- mice. Our results thus establish that PARP-1 acts as a survival factor for intestinal stem cells in vivo and suggest a functional link with early p53 and p21CIP1/WAF1 responses

A review on the immobilization of bromelain

This review shows the endeavors performed to prepare immobilized formulations of bromelain extract, usually from pineapple, and their use in diverse applications. This extract has a potent proteolytic component that is based on thiol proteases, which differ depending on the location on the fruit. Stem and fruit are the areas where higher activity is found. The edible origin of this enzyme is one of the features that determines the applications of the immobilized bromelain to a more significant degree. The enzyme has been immobilized on a wide diversity of supports via different strategies (covalent bonds, ion exchange), and also forming ex novo solids (nanoflowers, CLEAs, trapping in alginate beads, etc.). The use of preexisting nanoparticles as immobilization supports is relevant, as this facilitates one of the main applications of the immobilized enzyme, in therapeutic applications (as wound dressing and healing components, antibacterial or anticancer, mucus mobility control, etc.). A curiosity is the immobilization of this enzyme on spores of probiotic microorganisms via adsorption, in order to have a perfect in vivo compatibility. Other outstanding applications of the immobilized enzyme are in the stabilization of wine versus haze during storage, mainly when immobilized on chitosan. Curiously, the immobilized bromelain has been scarcely applied in the production of bioactive peptides.We gratefully recognize the financial support from Ministerio de Ciencia e Innovación and Agencia Estatal de Investigación (Spanish Government) and FEDER (PID2022-136535OB-I00). The authors gratefully acknowledge FAPESP (São Paulo Research Foundation) by research scholarship to DA (Grant No: 2023/01338-7). A.B.M. would like to thank the Generalitat Valenciana (PROMETEO Project CIPROM/2021/70) and the PID2021-123079OB-I00 project funded by MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”. for the financial support

Frequency of tuberculous and non-tuberculous mycobacteria in HIV infected patients from Bogota, Colombia

BACKGROUND: The prevalence of infections by Mycobacterium tuberculosis and non-tuberculous Mycobacterium species in the HIV-infected patient population in Colombia was uncertain despite some pilot studies. We determined the frequency of isolation of Mycobacterium tuberculosis and of non-tuberculous Mycobacterium species in diverse body fluids of HIV-infected patients in Bogota, Colombia. METHODS: Patients who attended the three major HIV/AIDS healthcare centres in Bogota were prospectively studied over a six month period. A total of 286 patients were enrolled, 20% of them were hospitalized at some point during the study. Sixty four percent (64%) were classified as stage C, 25% as stage B, and 11% as stage A (CDC staging system, 1993). A total of 1,622 clinical samples (mostly paired samples of blood, sputum, stool, and urine) were processed for acid-fast bacilli (AFB) stain and culture. RESULTS: Overall 43 of 1,622 cultures (2.6%) were positive for mycobacteria. Twenty-two sputum samples were positive. Four patients were diagnosed with M. tuberculosis (1.4%). All isolates of M. tuberculosis were sensitive to common anti-tuberculous drugs. M. avium was isolated in thirteen patients (4.5%), but only in three of them the cultures originated from blood. The other isolates were obtained from stool, urine or sputum samples. In three cases, direct AFB smears of blood were positive. Two patients presented simultaneously with M. tuberculosis and M. avium. CONCLUSIONS: Non-tuberculous Mycobacterium infections are frequent in HIV infected patients in Bogota. The diagnostic sensitivity for infection with tuberculous and non-tuberculous mycobacteria can be increased when diverse body fluids are processed from each patient

Periodogram Connectivity of EEG Signals for the Detection of Dyslexia

Electroencephalography (EEG) signals provide an important source of information of brain activity at different areas. This information can be used to diagnose brain disorders according to different activation patterns found in controls and patients. This acquisition technology can be also used to explore the neural basis of less evident learning disabilities such as Developmental Dyslexia (DD). DD is a specific difficulty in the acquisition of reading skills not related to mental age or inadequate schooling, whose prevalent is estimated between 5% and 12% of the population. In this paper we propose a method to extract discriminative features from EEG signals based on the relationship among the spectral density at each channel. This relationship is computed by means of different correlation measures, inferring connectivity-like markers that are eventually selected and classified by a linear support vector machine. The experiments performed shown AUC values up to 0.7, demonstrating the applicability of the proposed approach for objective DD diagnosis

Poly[ADP-Ribose] Polymerase-1 Expression Is Related To Cold Ischemia, Acute Tubular Necrosis, and Delayed Renal Function In Kidney Transplantation

Cold ischemia time especially impacts on outcomes of expanded-criteria donor (ECD) transplantation. Ischemia-reperfusion (IR) injury produces excessive poly[ADP-Ribose] Polymerase-1 (PARP-1) activation. The present study explored the hypothesis that increased tubular expression of PARP-1 contributes to delayed renal function in suboptimal ECD kidney allografts and in non-ECD allografts that develop posttransplant acute tubular necrosis (ATN)

Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity

Background: Environmental mycobacteria (EM) include species commonly found in various terrestrial and aquatic environments, encompassing animal and human pathogens in addition to saprophytes. Approximately 150 EM species can be separated into fast and slow growers based on sequence and copy number differences of their 16S rRNA genes. Cultivation methods are not appropriate for diversity studies; few studies have investigated EM diversity in soil despite their importance as potential reservoirs of pathogens and their hypothesized role in masking or blocking M. bovis BCG vaccine. Methods: We report here the development, optimization and validation of molecular assays targeting the 16S rRNA gene to assess diversity and prevalence of fast and slow growing EM in representative soils from semi tropical and temperate areas. New primer sets were designed also to target uniquely slow growing mycobacteria and used with PCR-DGGE, tag-encoded Titanium amplicon pyrosequencing and quantitative PCR. Results: PCR-DGGE and pyrosequencing provided a consensus of EM diversity; for example, a high abundance of pyrosequencing reads and DGGE bands corresponded to M. moriokaense, M. colombiense and M. riyadhense. As expected pyrosequencing provided more comprehensive information; additional prevalent species included M. chlorophenolicum, M. neglectum, M. gordonae, M. aemonae. Prevalence of the total Mycobacterium genus in the soil samples ranged from 2.3×107 to 2.7×108 gene targets g−1; slow growers prevalence from 2.9×105 to 1.2×107 cells g−1. Conclusions: This combined molecular approach enabled an unprecedented qualitative and quantitative assessment of EM across soil samples. Good concordance was found between methods and the bioinformatics analysis was validated by random resampling. Sequences from most pathogenic groups associated with slow growth were identified in extenso in all soils tested with a specific assay, allowing to unmask them from the Mycobacterium whole genus, in which, as minority members, they would have remained undetected