AESTRAC~Rifabutin is an antibiotic of the rifamycin class, which is particularly active against mycobacteria, including those that occur in AIDS patients. Because clinical use will include long-term therapy, an extensive battery of long-term toxicity studies was carried out by the oral route, including carcinogenicity studies. An interesting feature was the occurrence of multinucleated hepatocytes (MNHs) in the rat. In some instances, as many as 25 nuclei occurred in a single cell. Light microscopy revealed a large hepatocyte with normal eosinophilic staining. The multiple nuclei stained like those present in the surrounding normal cells. Electron microscopy showed no abnormalities of the nuclei and no cell membranes within the cytoplasm. The customary organelles were present. MNHs were dose-and sex-related, starting from 10 mg/kg/day and being more evident in males. They began to appear after 5 wk of treatment and persisted over long periods of recovery ( I 2 mo), without showing any tendency for cell proliferation. The life-span of MNHs was similar to that of normal hepatocytes. MNHs were present in the carcinogenicity study, but there was no increase in liver tumors.MNHs did not occur in mice or monkeys treated with rifabutin, nor did they occur in response to treatment with rifampin. The effect appears to be specific to the rat.Keywords. Liver; antibiotic . INTRODUC~ION Rifabutin is an antibiotic of the rifamycin class, which is particularly active against mycobacteria including those that occur in AIDS patients. Because clinical use will include long-term therapy, an extensive battery of long-term toxicity studies was carried out by the oral route, including carcinogenicity studies.At the beginning of the customary safety experiments in the rat, the presence in the liver of multinucleated hepatocytes (MNHs) was discovered. In this article, we report the details of our extensive investigation of the phenomenon. MATERIALS AND MITHODSThe customary toxicity studies for safety evaluation were carried out in rats, mice, and monkeys.The majority of the studies, particularly those of rats, were carried out at Farmitalia Carlo Erba, whereas a proportion of those in mice and monkeys were performed under the supervision of Farmitalia Carlo Erba in independent contract laboratories. Materials, methods, and results are to be reported separately (4). The details of the studies are given in Table I. In these, the customary investigations were performed including the recording of body weight, clinical signs, ophthalmoscopy, food and water consumption, hematology, and clinical biochemistry. At termination, a full necropsy was carried out, the customary organs were weighed, and pumerous tissues were examined for histopathologYFor these studies, rifabutin of pharmaceutical grade and 96.48% purity was obtained from the Department , . .... of R&D/Galenics, Farmitalia (Milan) and, similarly, rifampin of 94.6% purity was supplied by SPA (Milan).Because MNHs were seen to occur in the liver of rats with evidence of a dose-related e...
Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our ∼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.
Megasynthase enzymes such as type I modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) play a central role in microbial chemical warfare because they can evolve rapidly by shuffling parts (catalytic domains) to produce novel chemicals. If we can understand the design rules to reshuffle these parts, PKSs and NRPSs will provide a systematic and modular way to synthesize millions of molecules including pharmaceuticals, biomaterials, and biofuels. However, PKS and NRPS engineering remains difficult due to a limited understanding of the determinants of PKS and NRPS fold and function. We developed ClusterCAD to streamline and simplify the process of designing and testing engineered PKS variants. Here, we present the highly improved ClusterCAD 2.0 release, available at https://clustercad.jbei.org. ClusterCAD 2.0 boasts support for PKS-NRPS hybrid and NRPS clusters in addition to PKS clusters; a vastly enlarged database of curated PKS, PKS-NRPS hybrid, and NRPS clusters; a diverse set of chemical ‘starters’ and loading modules; the new Domain Architecture Cluster Search Tool; and an offline Jupyter Notebook workspace, among other improvements. Together these features massively expand the chemical space that can be accessed by enzymes engineered with ClusterCAD.
Foldy is a cloud-based application that allows non-expert scientists to easily access and utilize advanced AI-based structural biology tools, including AlphaFold and DiffDock. Built on Kubernetes, it can be deployed by universities, departments, and labs without requiring hardware resources, but can also be configured to utilize available computers. Foldy enables scientists to predict the structure of proteins and complexes up to 3000 amino acids, visualize Pfam annotations, and dock ligands with AutoDock Vina and DiffDock. Our manuscript describes the user interface and deployment considerations of Foldy, as well as some of our applications. By democratizing access to sophisticated AI-based tools, Foldy can facilitate life science research and promote the wider adoption of structural bioinformatics tools. Our work demonstrates that even the most advanced tools can be made accessible to a broad audience through user-friendly platforms like Foldy, and we believe it will be a valuable resource for researchers across scientific disciplines. The public structures available on the Lawrence Berkeley Labs Foldy deployment can be viewed at https://foldy.lbl.gov.
Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, the resulting PKS hybrids typically have reduced catalytic activities and are often insoluble due to misfolding. Here, we have developed a fluorescence-based biosensor method for detecting engineered PKSs with high solubility. The biosensor has been used to sort through PKS hybrids that had acyltransferase (AT) domains from other PKSs exchanged for the native AT with randomly assigned linker junctions. Importantly, we observed a significant correlation between activity and solubility. Evaluation of highly soluble mutants in vitro revealed new boundaries for AT domain exchanges that give a wild-type level of catalytic activity. Together, we have successfully developed an experimentally validated high-throughput method to efficiently screen active engineered PKSs that produce target molecules.
In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For the AraC-family regulators (AFRs) represented in this data, we posited AFR/promoter/inducer groupings. Eleven promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in E. coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled further diversification of these inducible plasmids.
Novaccta S p.A L'iale Picmonlc 66 2001 3 h1ngcnt;i. Italy Demetrio Pitea Institutc 01' Physical Chcmistry University of Milnn Via C. (iolgi 1' 9 20133 Milano. It;ily A merhod of color control of bulk-d.wd cellulose acefate is described, based on the tran,smiftani,~i spectrum of the dope. (.onfro1 can be ej'ected only if the spectruwr is recordedfi-om thick samples which ampl$y the absorption from stnall amounts of impurities that later cause unacceptable color dij"erences on the yarn. The calculation of the reflec.tunce spectrum, and hence the color, ofthe yarn f r o m the transmitfanre spectrum of f h e dope by Kubelka-Munk theory i s not satisfactory due to Jhint f u rhidity (?f'the "bright" dope. The rtlflectance spectrum of the yarn can be calculated from a series expunsion ofthe experimrntal Lambert law ubsorprion cocdjirient with very safisf&tory results. A fully computer-nmiuged colorcontrol method i s employed .successfully in industrial practice.
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