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Estrogen receptor beta as a novel target of androgen receptor action in breast cancer cell lines.

INTRODUCTION: The two isoforms of estrogen receptor (ER) alpha and beta play opposite roles in regulating proliferation and differentiation … t cancer cell lines using real-time PCR, Western blotting and small interfering RNA (siRNA) assays. Mutagenesis studies, electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were performed to assess the effects of mibolerone/AR on ER-beta promoter activity and binding. RESULTS: In this study, we demonstrate that mibolerone, a …

Analysis of the interaction of tarantula toxin Jingzhaotoxin-III (β-TRTX-Cj1α) with the voltage sensor of Kv2.1 uncovers the molecular basis for cross-activities on Kv2.1 and Nav1.5 channels.

Animal venoms contain a fascinating array of divergent peptide toxins that have cross-activities on different types of voltage-gated ion cha … .4, Kv3.1, and Kv4.1-4.3), whereas it significantly inhibited Kv2.1 with an IC50 of 0.71 ± 0.01 μM. Mutagenesis and modeling data suggested that JZTX-III docks at the Kv2.1 voltage-sensor paddle. Alanine replacement of Phe274, Lys280, Ser281, Leu283, Gln284, and Val288 could decrease JZTX-III affinity by 7-, 9-, 34-, 12-, 9-, and 7-fold, respectively. Among t …

The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli clone.

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstr … pin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative o …

Regulation of insulin gene transcription by the immediate-early growth response gene Egr-1.

Changes in extracellular glucose levels regulate the expression of the immediate-early response gene and zinc finger transcription factor ea … essed insulin gene expression. Egr-1 did not directly interact with insulin promoter sequences, and mutagenesis of a potential G box recognition sequence for Egr-1 did not impair the Egr-1 responsiveness of the insulin promoter, suggesting that regulation of insulin gene expression by Egr-1 is probably mediated through additional transcription factors. Overex …

Amino acid 305 determines catalytic center accessibility in CYP3A4.

Site-directed mutagenesis has been used to replace alanine 305 with phenylalanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and testosterone metabolism have been determined for both mutants and wild-type CYP3A4. The A305F mutation abolished diazepam oxidase activity and reduced the S(50) and V(max) for erythromycin N-demethylase activity from 17 to 10 microM and from 3.2 to 1.2 pmol product/min/pmol P450, respectively. The V(max) for testosterone 6beta-hydroxylase activity was also significantly reduce…

Protocol for non-invasive delivery of CRISPR RNPs via virus-like particles for mouse model generation.

CRISPR-virus-like particle (VLP)-induced targeted mutagenesis (CRISPR-VIM) enables genome editing in mouse embryos through non-invasive delivery of CRISPR ribonucleoproteins (RNPs) via VLPs, eliminating the need for physical manipulation and specialized expertise. We detail protocols for VLP production, titration, and treatment for diverse genome edits. This protocol is compatible with zygotes and in vitro fertilization (IVF)-derived embryos via simple co-culture, facilitating high-efficiency and heritable mutations with minimized off-target effects, independent of specialized equipment and co…

Discovery of Zika virus host dependency factors in trophoblasts using CRISPR/Cas9 screening.

Emerging mosquito-borne RNA viruses cause massive health complications worldwide. The Zika virus (ZIKV), in particular, has spread dramatically since 2007 and has provoked epidemics in the Americas and the South Pacific. The lack of antiviral therapy and vaccination has focused research on the investigation of ZIKV-host interactions, in order to understand underlying molecular infection mechanisms. We have established an approach for the analysis of ZIKV host dependency factors in a human trophoblast cell line and applied genome-wide CRISPR/Cas9 knockout mutagenesis. The presented method is es…

Структурная основа для гомотипного синтеза хламидиальных интеграций с помощью NASER-подобных белковых IncA.

Многие внутриклеточные бактерии, включая хламидию, создают паразитически привязанную к мембране органеллу внутри принимающей клетки, которая необходима для выживания бактерий. Chlamidia trachomatis образует вложения, которые украшены плохо описываемыми мембранными белками, известными как Inc. Прототипичная инк., называемая IncA, повышает патогенность хламидии, пропагандируя гомотипное слияние интеграций и делясь структурным и функциональным сходством с эукариотической СНСРЕ. Здесь мы представляем атомную структуру цитоплазмической области IncA, которая обнаруживает неканоническую четырёхлексну

Совпадение в аннотации оригинала
Many intracellular bacteria, including Chlamydia, establish a parasitic membrane-bound organelle inside the host cell that is essential for … of the cytoplasmic domain of IncA, which reveals a non-canonical four-helix bundle. Structure-based mutagenesis, molecular dynamics simulation, and functional cellular assays identify an intramolecular clamp that is essential for IncA-mediated homotypic membrane fusion during infection.

A model of BmK CT in inhibiting glioma cell migration via matrix metalloproteinase-2 from experimental and molecular dynamics simulation study.

The significance of BmK CT, a key chlorotoxin-like peptide isolated from the scorpion venom of Buthus martensii Karsch, is a novel blocker of the chloride ion channel and matrix metalloproteinase-2 (MMP-2). Site-directed mutagenesis of BmK CT, wound healing assay, gelatin zymography assay and computational simulation highlight the importance of electrostatic contribution to BmK CT-MMP-2 catalytic domain complex and a model of BmK CT-MMP-2 catalytic domain complex is therefore proposed. This is the first documentation of the structural mechanism of in the inhibition of glioma cell migration by …

The solution structure of coronaviral stem-loop 2 (SL2) reveals a canonical CUYG tetraloop fold.

The transcription and replication of the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is regulated by specific viral genome sequences within 5'- and 3'-untranslated regions (5'-UTR and 3'-UTR). Here we report the solution structure of 5'-UTR derived stem-loop 2 (SL2) of SARS-CoV determined by NMR spectroscopy. The highly conserved pentaloop of SL2 is stacked on 5-bp stem and adopts a canonical CUYG tetraloop fold with the 3' nucleotide (U51) flipped out of the stack. The significance of this structure in the context of a previous mutagenesis analysis of SL2 function in repli…

The mouse Fused toes (Ft) mutation is the result of a 1.6-Mb deletion including the entire Iroquois B gene cluster.

As a result of transgenic insertional mutagenesis, heterozygous Fused toes (Ft) mice display a syndactyly of forelimbs and a thymic hyperplasia. Homozygous Ft/Ft embryos die in midgestation, exhibiting a deformation of craniofacial structures, a syndactyly and a polydactyly of fore- and hindlimbs, a disorganization of the ventral spinal cord, and defects in left-right axis formation. Here we report on our structural analyses of the Ft mutation. We established a physical as well as a gene map of the Ft locus, showing that the transgene integration resulted in a deletion of 1.6 Mb of genomic seq…

Structural basis of the interaction of the pyelonephritic E. coli adhesin to its human kidney receptor.

PapG is the adhesin at the tip of the P pilus that mediates attachment of uropathogenic Escherichia coli to the uroepithelium of the human kidney. The human specific allele of PapG binds to globoside (GbO4), which consists of the tetrasaccharide GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc linked to ceramide. Here, we present the crystal structures of a binary complex of the PapG receptor binding domain bound to GbO4 as well as the unbound form of the adhesin. The biological importance of each of the residues involved in binding was investigated by site-directed mutagenesis. These studies provi…

Thermosensitive mutants of the MPTP and hPTP1B protein tyrosine phosphatases: isolation and structural analysis.

A PCR-based random mutagenesis procedure was employed to identify several thermosensitive mutants of the MPTP enzyme, the murine homologue of the human T-cell PTPase and rat PTP-S enzymes. Four mutants with varying degrees of thermosensitivity were characterized for their thermostability and refolding properties following incubation at the nonpermissive temperature. Structure analysis of these mutations based on the hPTP1B co-ordinate structure demonstrates a clear relationship between the position of each mutated amino acid relative to the catalytic cysteine residue and their thermostability.…

Construction of Salmonella Transposon Mutant Library.

One of the main challenges in understanding Salmonella biology lies in dissecting the genetic basis of its survival, pathogenicity, and antimicrobial resistance. Here, we describe the construction of a high-density transposon mutant library in Salmonella. Using a mariner-based transposon system delivered through conjugation, we generated a library comprising more than 1 × 105 independent mutants, covering the majority of nonessential genes. This library enables a valuable resource for large-scale functional genomics studies, including the identification of essential genes, virulence determinat

Engineering and immobilization of imine reductase enable chemoenzymatic synthesis of SLC6A19 inhibitor JNT-517.

Enzymes represent renewable biological resources, and their employment as industrial biocatalysts not only enhances production efficiency but also mitigates environmental pollution associated with excessive organic solvent usage. (R)-N-Cyclopropylpiperidin-3-amine serves as a crucial pharmaceutical intermediate, traditionally produced via chiral resolution. Imine reductases (IREDs) present an atom-efficient alternative by directly catalyzing the reductive amination of prochiral ketones with amines to yield chiral alkylated amines. Through site-directed mutagenesis of IR-G36-M5, we developed th…

Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters.

The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames tes

Development of Genetically Encoded Biosensors for Reporting the Methyltransferase-Dependent Biosynthesis of Semisynthetic Macrolide Antibiotics.

Clarithromycin is an improved semisynthetic analogue of the naturally occurring macrolide, erythromycin. The subtle modification of a methyl group on the C-6 hydroxyl group endows the molecule with improved acid stability and results in a clinically useful antibiotic. Here, we show that the effector specificity of the biosensor protein, MphR, can be evolved to selectively recognize clarithromycin and therefore report on the production of this molecule in vivo. In addition, a crystal structure of the evolved variant reveals the molecular basis for selectivity and provides a guide for the evolut

A novel 13 base pair insertion in the sonic hedgehog ZRS limb enhancer (ZRS/LMBR1) causes preaxial polydactyly with triphalangeal thumb.

Mutations in the Sonic hedgehog limb enhancer, the zone of polarizing activity regulatory sequence (ZRS, located within the gene LMBR1), commonly called the ZRS), cause limb malformations. In humans, three classes of mutations have been proposed based on the limb phenotype; single base changes throughout the region cause preaxial polydactyly (PPD), single base changes at one specific site cause Werner mesomelic syndrome, and large duplications cause polysyndactyly. This study presents a novel mutation-a small insertion. In a Swedish family with autosomal-dominant PPD, we found a 13 base pair i

Новый принцип смягчения последствий для разработки вакцин нового поколения с живым ароматом.

К роду Флавивирус относится ряд важных человеческих патогенов, которые создают серьезные проблемы со здоровьем в крупных регионах мира. Появляющиеся в новых географических регионах ароматизаторы (например, вирус Западного Нила в Северной Америке) и стремительные социально-экономические изменения произошли во многих развивающихся странах, где такие флавивирусы, как вирус денге и японский энцефалит и эндемический вирус, требуют разработки новых вакцин против этих заболеваний. Используя в качестве модели вирус энцефалита тика, мы разработали новый метод создания смягченных ароматических штаммов,