The mast cells of the mammalian central nervous system V. The effect of compound 48|80 on the neurolipomastocytoid cells and related areas of the CNS: Early changes

Summary The response of the neurolipomastocytoid cells (NLMs) and elements in their vicinity within the central nervous system of various animal species was studied following injection of the animals with the specific mast cell (MC)-discharger compound 48/80. The observed alterations were grouped into those occurring early (0–21 days) and later (up to 18 months). In the present report, only the acute changes are described, light and electron microscopically.Most experimental animals developed prostration, scratching, acral-type reaction, signs of respiratory distress and salivation, and, in the monkey, uncontrollable somnolence. Within about 2 weeks after the injection some animals (especially guinea pigs) manifested various degrees of limb paralysis. The NLMs, like MCs outside the CNS, responded to injection by various degrees of degranulation, vacuolation, marked variation in granule size, apparent cell loss and sometimes an increase in number. Electron microscopically, particulate breakdown products of the granules of the NLMs appeared in the cytoplasm; occasionally there was suggestive evidence that they had passed inward across the vessel wall to reach the lumen, and also outward through the outermost basal lamina. Perivascular astrocytic feet showed swelling and vacuolation shortly after the injection, which was followed by evidence of gliosis and later scarring; occasionally, alterations in the mitochondria were observed. In the spinal cord of the guinea pig, capillary neoformation was observed with endothelial cells and adjacent NLMs taking up tritiated thymidine.The discussion centers on the partial similarity of response to compound 48/80 of the NLMs to that of MCs outside the CNS, and the probable involvement of NLM-damage in the parenchymal changes.The preliminary portion of this work was done with T. Wiedman at Ames Research Center, Moffett Field, California; a short communication has been published (Ibrahim 1970), and part of the study was presented at the Annual Meeting of the American Association of Anatomists (Ibrahim et al. 1976)Supported by a grant from the Incentive Plan of the Medical School, American University of Beirut, Lebanon, by Biomedical Research support Grant RR 05373 from the Biomedical Research Support Branch, Division of Research Facilities and Resources, National Institutes of Health, and by Research Grant No. MA 004 from the College of Graduate Studies, University of Kuwait     

A simple reversed phase high-performance liquid chromatography method for polysorbate 80 quantitation in monoclonal antibody drug products

In this paper, we discuss an improved high-performance liquid chromatography (HPLC) method for the quantitation of polysorbate 80 (polyoxyethylenesorbitan monooleate), a commonly used stabilizing excipient in therapeutic drug solutions. This method is performed by quantitation of oleic acid, a hydrolysis product of polysorbate 80. Using base hydrolysis, polysorbate 80 releases the oleic acid at a 1:1 molar ratio. The oleic acid can then be separated from other polysorbate 80 hydrolysis products and matrices using reversed phase HPLC. The oleic acid is monitored without derivatization using the absorbance at 195 nm. The method was validated and also shown to be accurate for the quantitation of polysorbate 80 in a high protein concentration monoclonal antibody drug product. For the measured polysorbate 80 concentrations, the repeatability was less than 6.2% relative standard deviation of the mean (% RSD) with the day-to-day intermediate precision being less than 8.2% RSD. The accuracy of the oleic acid quantitation averaged 94–109% in different IgG₁ and IgG₄ drug solutions with variable polysorbate 80 concentrations. In this study, polyoxyethylene, a by-product of the polysorbate 80 hydrolysis was also identified. This peak was not identified by previous methods and also increased proportionally to the polysorbate 80 concentration. We have developed and qualified a method which uses common equipment found in most laboratories and is usable over a range of monoclonal antibody subclasses and protein concentrations.     

CHIP and BAP1 Act in Concert to Regulate INO80 Ubiquitination and Stability for DNA Replication

The INO80 chromatin remodeling complex has roles in many essential cellular processes, including DNA replication. However, the mechanisms that regulate INO80 in these processes remain largely unknown. We previously reported that the stability of Ino80, the catalytic ATPase subunit of INO80, is regulated by the ubiquitin proteasome system and that BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, stabilizes Ino80 via deubiquitination and promotes replication fork progression. However, the E3 ubiquitin ligase that targets Ino80 for proteasomal degradation was unknown. Here, we identified the C-terminus of Hsp70-interacting protein (CHIP), the E3 ubiquitin ligase that functions in cooperation with Hsp70, as an Ino80-interacting protein. CHIP polyubiquitinates Ino80 in a manner dependent on Hsp70. Contrary to our expectation that CHIP degrades Ino80, CHIP instead stabilizes Ino80 by extending its half-life. The data suggest that CHIP stabilizes Ino80 by inhibiting degradative ubiquitination. We also show that CHIP works together with BAP1 to enhance the stabilization of Ino80, leading to its chromatin binding. Interestingly, both depletion and overexpression of CHIP compromise replication fork progression with little effect on fork stalling, as similarly observed for BAP1 and Ino80, indicating that an optimal cellular level of Ino80 is important for replication fork speed but not for replication stress suppression. This work therefore idenitifes CHIP as an E3 ubiquitin ligase that stabilizes Ino80 via nondegradative ubiquitination and suggests that CHIP and BAP1 act in concert to regulate Ino80 ubiquitination to fine-tune its stability for efficient DNA replication.     

A novel small molecule inhibitor of the DNA repair protein Ku70/80

Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PK_CS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.     

Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes

The frequency of targeted gene disruption via homologous recombination is low in the clinically important dermatophyte, Trichophyton mentagrophytes . The Ku genes, Ku70 and Ku80 , encode key components of the nonhomologous end-joining pathway involved in DNA double-strand break repair. Their deletion increases the homologous recombination frequency, facilitating targeted gene disruption. To improve the homologous recombination frequency in T. mentagrophytes , the Ku80 ortholog was inactivated. The nucleotide sequence of the Ku80 locus containing a 2788-bp ORF encoding a predicted product of 728 amino acids was identified, and designated as TmKu80 . The predicted TmKu80 product showed a high degree of amino acid sequence similarity to known fungal Ku80 proteins. Ku80 disruption mutant strains of T. mentagrophytes were constructed by Agrobacterium tumefaciens -mediated genetic transformation. The average homologous recombination frequency was 73.3 ± 25.2% for the areA/nit-2 -like nitrogen regulatory gene ( tnr ) in Ku80⁻ mutants, about 33-fold higher than that in wild-type controls. A high frequency ( c . 67%) was also obtained for the Tri m4 gene encoding a putative serine protease. Ku80 ⁻ mutant strains will be useful for large-scale reverse genetics studies of dermatophytes, including T. mentagrophytes , providing valuable information on the basic mechanisms of host invasion.     

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.     

Developmental and housekeeping transcriptional programs in Drosophila require distinct chromatin remodelers

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A hexasome is the preferred substrate for the INO80 chromatin remodeling complex,allowing versatility of function

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