STAT3 Polymorphism Associates With mTOR Inhibitor-Induced Interstitial Lung Disease in Patients With Renal Cell Carcinoma.

We evaluated the association of signal transducer and activator of transcription 3 (STAT3) polymorphisms with the incidence of mammalian target of rapamycin (mTOR) inhibitor-induced interstitial lung disease (ILD) in patients with renal cell carcinoma (RCC). We also used lung-derived cell lines to investigate the mechanisms of this association. Japanese patients with metastatic RCC who were treated with mTOR inhibitors were genotyped for the STAT3 polymorphism, rs4796793 (1697C/G). We evaluated the association of the STAT3 genotype with the incidence of ILD and therapeutic outcome. In the 57 patients included in the primary analysis, the ILD rate within 140 days was significantly higher in patients with the GG genotype compared with those with other genotypes (77.8% vs. 23.1%, odds ratio=11.67, 95% confidential interval=3.0644.46). There were no significant differences in progression-free survival or time-to-treatment failure between the patients with the GG genotype and those with other genotypes. An in vitro study demonstrated that some lung-derived cell lines carrying the GG genotype exhibited an increase in the expression of mesenchymal markers, such as fibronectin, N-cadherin, and vimentin, and decreases in E-cadherin, which is an epithelial marker associated with exposure to everolimus, although STAT3 expression and activity were not related to the genotype. In conclusion, the GG genotype of the STAT3 rs4796793 polymorphism increases the risk of mTOR inhibitor-induced ILD, supporting its use as a predictive marker for RCC.

Dynamic changes in the interchromosomal interaction of early histone gene loci during development of sea urchin.

The nuclear positioning and chromatin dynamics of eukaryotic genes are closely related to the regulation of gene expression, but they have not been well examined during early development, which is accompanied by rapid cell cycle progression and dynamic changes in nuclear organization, such as nuclear size and chromatin constitution. In this study, we focused on the early development of the sea urchin Hemicentrotus pulcherrimus and performed three-dimensional fluorescence in situ hybridization of gene loci encoding early histones (one of the types of histone in sea urchin). There are two non-allelic early histone gene loci per sea urchin genome. We found that during the morula stage, when the early histone gene expression levels are at their maximum, interchromosomal interactions were often formed between the early histone gene loci on separate chromosomes and that the gene loci were directed to locate to more interior positions. Furthermore, these interactions were associated with the active transcription of the early histone genes. Thus, such dynamic interchromosomal interactions may contribute to the efficient synthesis of early histone mRNA during the morula stage of sea urchin development.

Fusion protein analysis reveals the precise regulation between Hsp70 and Hsp100 during protein disaggregation.

ClpB, a bacterial Hsp100, is a ring-shaped AAA+ chaperone that can reactivate aggregated proteins in cooperation with DnaK, a bacterial Hsp70, and its co-factors. ClpB subunits comprise two AAA+ modules with an interstitial rod-shaped M-domain. The M-domain regulates ClpB ATPase activity and interacts directly with the DnaK nucleotide-binding domain (NBD). Here, to clarify how these functions contribute to the disaggregation process, we constructed ClpB, DnaK, and aggregated YFP fusion proteins in various combinations. Notably, i) DnaK activates ClpB only when the DnaK substrate-binding domain (SBD) is in the closed conformation, affording high DnaK-peptide affinity; ii) although NBD alone can activate ClpB, SBD is required for disaggregation; and iii) tethering aggregated proteins to the activated ClpB obviates SBD requirements. These results indicate that DnaK activates ClpB only when the SBD tightly holds aggregated proteins adjacent to ClpB for effective disaggregation.

Opaliferin, a new polyketide from cultures of entomopathogenic fungus Cordyceps sp. NBRC 106954.

Opaliferin, a polyketide with a unique partial structure in which a cyclopentanone and tetrahydrofuran were connected with an external double bond, was isolated from the insect pathogenic fungus Cordyceps sp. NBRC 106954. The structure and relative configuration of opaliferin were determined by spectroscopic analysis and X-ray crystallography. The absolute configuration was established by anomalous dispersion effects in X-ray diffraction measurements on the crystal of di(p-bromobenzoyl) ester of opaliferin. A plausible biosynthetic pathway for opaliferin is proposed.

In vitro antitrypanosomal activity of the cyclodepsipeptides, cardinalisamides A-C, from the insect pathogenic fungus Cordyceps cardinalis NBRC 103832.

During the search for new antitrypanosomal drug leads, three new antitrypanosomal compounds, cardinalisamides A-C (1-3), were isolated from cultures of the insect pathogenic fungus Cordyceps cardinalis NBRC 103832. Their structures were elucidated using MS analyses and extensive 2D-heteronuclear NMR. The absolute configurations of 1-3 were addressed by chemical degradation and Marfey's analysis. 1-3 showed in vitro antitrypanosomal activity against Trypanosoma brucei brucei with IC50 values of 8.56, 8.65 and 8.63 µg ml(-1), respectively.

Elucidation of the active conformation of vancomycin dimers with antibacterial activity against vancomycin-resistant bacteria.

Covalently linked vancomycin dimers have attracted a great deal of attention among researchers because of their enhanced antibacterial activity against vancomycin-resistant strains. However, the lack of a clear insight into the mechanisms of action of these dimers hampers rational optimization of their antibacterial potency. Here, we describe the synthesis and antibacterial activity of novel vancomycin dimers with a constrained molecular conformation achieved by two tethers between vancomycin units. Conformational restriction is a useful strategy for studying the relationship between the molecular topology and biological activity of compounds. In this study, two vancomycin units were linked at three distinct positions of the glycopeptide (vancosamine residue (V), C terminus (C), and N terminus (N)) to form two types of novel vancomycin cyclic dimers. Active NC-VV-linked dimers with a stable conformation as indicated by molecular mechanics calculations selectively suppressed the peptidoglycan polymerization reaction of vancomycin-resistant Staphylococcus aureus in vitro. In addition, double-disk diffusion tests indicated that the antibacterial activity of these dimers against vancomycin-resistant enterococci might arise from the inhibition of enzymes responsible for peptidoglycan polymerization. These findings provide a new insight into the biological targets of vancomycin dimers and the conformational requirements for efficient antibacterial activity against vancomycin-resistant strains.

Substitutions of coenzyme-binding, nonpolar residues improve the low-temperature activity of thermophilic dehydrogenases.

Although enzymes of thermophilic organisms are often very resistant to thermal denaturation, they are usually less active than their mesophilic or psychrophilic homologues at moderate or low temperatures. To explore the structural features that would improve the activity of a thermophilic enzyme at less than optimal temperatures, we randomly mutated the DNA of single-site mutants of the thermostable Thermus thermophilus 3-isopropylmalate dehydrogenase that already had improved low-temperature activity and selected for additional improved low-temperature activity. A mutant (Ile279 → Val) with improved low-temperature activity contained a residue that directly interacts with the adenine of the coenzyme NAD(+), suggesting that modulation of the coenzyme-binding pocket's volume can enhance low-temperature activity. This idea was further supported by a saturation mutagenesis study of the two codons of two other residues that interact with the adenine. Furthermore, a similar type of amino acid substitution also improved the catalytic efficiency of another thermophilic dehydrogenase, T. thermophilus lactate dehydrogenase. Steady-state kinetic experiments showed that the mutations all favorably affected the catalytic turnover numbers. Thermal stability measurements demonstrated that the mutants remain very resistant to heat. Calculation of the energetic contributions to catalysis indicated that the increased turnover numbers are the result of destabilized enzyme-substrate-coenzyme complexes. Therefore, small changes in the side chain volumes of coenzyme-binding residues improved the catalytic efficiencies of two thermophilic dehydrogenases while preserving their high thermal stabilities and may be a way to improve low-temperature activities of dehydrogenases in general.

Synthesis of rigidly-linked vancomycin dimers and their in vivo efficacy against resistant bacteria.

A novel and efficient avenue for the preparation of dimeric vancomycins is described, and the dimers exhibited excellent antibacterial activities in the murine infection model.