Design and synthesis of 4-acetoxypentanamide derivatives of spliceostatin A and their biological evaluation towards prostate cancer treatment.

We designed and synthesized novel 4-acetoxypentanamide derivatives of spliceostatin A, whose 4-acetoxypentenamide moiety is reduced (7), isomerized (8), or substituted with methyl at the α-position (9). The results of biological evaluation against AR-V7 and the docking analysis of each derivative suggest that the geometry of the 4-acetoxypentenamide moiety of spliceostatin A is important for its biological activity.

Combining mRNA with PBS and calcium ions improves the efficiency of the transfection of mRNA into tumors.

mRNA is a promising modality for expressing a protein in vivo. Drug delivery systems are required for the efficient transfection of mRNA into cells. In this study, we evaluated several drug delivery systems for transfecting mRNA into tumors. A lipid nanoparticle delivered mRNA to the draining lymph nodes and liver, even by intratumoral injection. A liposome-based system did not consistently provide mRNA for different types of tumor cells. We found that PBS introduced mRNA into several tumors, and calcium ions enhanced the efficiency, particularly in male mice. The circular dichroism spectrometer suggested a structural change in mRNA in PBS. Transmission electron microscopy revealed that calcium ions promoted the formation of mRNA nanoparticles in PBS. Transfection of mRNAs coding OX40-ligand, interleukin (IL)-36γ, and IL-23 by PBS + calcium ions attenuated tumor growth. Our results indicate that combining PBS with calcium ions promotes the transfection of mRNA into tumors. These data provide information for the development of methods for transfection of mRNA for cancer therapy.

Thermosensing function of the Escherichia coli redox sensor Aer.

Escherichia coli chemoreceptors can sense changes in temperature for thermotaxis. Here we found that the aerotaxis transducer Aer, a homolog of chemoreceptors lacking a periplasmic domain, mediates thermoresponses. We propose that thermosensing by the chemoreceptors is a general attribute of their highly conserved cytoplasmic domain (or their less conserved transmembrane domain).

Four Japanese Patients with Congenital Nephrogenic Diabetes Insipidus due to the AVPR2 Mutations.

Almost 90% of nephrogenic diabetes insipidus (NDI) is caused by mutations in the arginine vasopressin receptor 2 gene (AVPR2) on the X chromosome. Herein, we reported clinical and biochemical parameters in four cases of three unrelated Japanese families and analyzed the status of the AVPR2. Two of the four patients had poor weight gain. However, in the male and female sibling cases, neither had poor weight gain while toddlers, but in the male sibling, episodes of recurrent fever, polyuria, and polydipsia led to the diagnosis of NDI at 4 years of age. Analysis of AVPR2 identified two nonsense mutations (c.299_300insA; p.K100KfsX91 and c.296G > A; p.W99X) and one missense mutation (c.316C > T; p.R106C). These mutations were previously reported. The patient with c.316C > T; p.R106C had milder symptoms consistent with previous reports. Of the familial cases, the sister was diagnosed as having NDI, but a skewed X-inactivation pattern in her peripheral blood lymphocytes was not identified. In conclusion, our study expands the spectrum of phenotypes and characterized mutations in AVPR2 in NDI.

The role of polar localization in the function of an essential Caulobacter crescentus tyrosine kinase.

DivL is an essential tyrosine kinase in Caulobacter crescentus that controls an early step in the cell division cycle. We show here that DivL dynamically localizes to the stalk-distal cell pole and less frequently to the stalked cell pole during the S-phase. The kinase is subsequently released from the cell poles late in division and remains dispersed in the newly divided progeny stalk and swarmer cells. Mutational analysis of DivL in a DivL-GFP fusion protein demonstrated that the extreme C-terminus and residues in the conserved four-helix bundle, which is the phosphorylation-dimerization domain, are important for localization. We speculate that the four-helix bundle of the core catalytic domain may serve as a recognition site for the "localization machinery". Unexpectedly, a DivL protein with mutations in the C-terminal localization sequence, and an intact catalytic domain, efficiently complemented a divL null mutation. Thus, subcellular localization of DivL is not essential to its function in cell division regulation. Regulation of cell division by DivL does, however, depend on its localization in the cell membrane.

The core dimerization domains of histidine kinases contain recognition specificity for the cognate response regulator.

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.

Protein sequences and cellular factors required for polar localization of a histidine kinase in Caulobacter crescentus.

The Caulobacter crescentus sensor kinase DivJ is required for an early cell division step and localizes at the base of the newly formed stalk during the G1-to-S-phase transition when the protein is synthesized. To identify sequences within DivJ that are required for polar localization, we examined the ability of mutagenized DivJ sequences to direct localization of the green fluorescent protein. The effects of overlapping C-terminal deletions of DivJ established that the N-terminal 326 residues, which do not contain the kinase catalytic domain, are sufficient for polar localization of the fusion protein. Internal deletions mapped a shorter sequence between residues 251 and 312 of the cytoplasmic linker that are required for efficient localization of this sensor kinase. PleC kinase mutants, which are blocked in the swarmer-to-stalked-cell transition and form flagellated, nonmotile cells, also fail to localize DivJ. To dissect the cellular factors involved in establishing subcellular polarity, we have examined DivJ localization in a pleC mutant suppressed by the sokA301 allele of ctrA and in a pleD mutant, both of which display a supermotile, stalkless phenotype. The observation that these Mot(+) strains localize DivJ to a single cell pole indicate that localization may be closely coupled to the gain of motility and that normal stalk formation is not required. We have also observed, however, that filamentous parC mutant cells, which are defective in DNA segregation and the completion of cell separation, are motile and still fail to localize DivJ to the new cell pole. These results suggest that formation of new sites for DivJ localization depends on events associated with the completion of cell separation as well as the gain of motility. Analysis of PleC and PleD mutants also provides insights into the function of the His-Asp proteins in cell cycle regulation. Thus, the ability of the sokA301 allele of ctrA to bypass the nonmotile phenotype of the pleC null mutation provides evidence that the PleC kinase controls cell motility by initiating a signal transduction pathway regulating activity of the global response regulator CtrA. Analysis of the pleD mutant cell cycle demonstrates that disruption of the swarmer-to-stalked-cell developmental sequence does not affect the asymmetric organization of the Caulobacter cell cycle.

Characterization and crystallization of DivK, an essential response regulator for cell division and differentiation in Caulobacter crescentus.

DivK is an essential response regulator involved in the complex signal transduction network required for cell division and cell differentiation in Caulobacter crescentus. Small-angle X-ray scattering analysis was valuable for obtaining single crystals of the DivK recombinant protein. These crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 37.2, b = 40.5, c = 67.1 A and diffract beyond 1.6 A on a synchrotron beamline.

Crystallographic and biochemical studies of DivK reveal novel features of an essential response regulator in Caulobacter crescentus.

DivK is an essential response regulator in the Gram-negative bacterium Caulobacter crescentus and functions in a complex phosphorelay system that precisely controls the sequence of developmental events during the cell division cycle. Structure determinations of this single domain response regulator at different pH values demonstrated that the five-stranded alpha/beta fold of the DivK protein is fully defined only at acidic pH. The crystal structures of the apoprotein and of metal-bound DivK complexes at higher pH values revealed a synergistic pH- and cation binding-induced flexibility of the beta4-alpha4 loop and of the alpha4 helix. This motion increases the solvent accessibility of the single cysteine residue in the protein. Solution state studies demonstrated a 200-fold pH-dependent increase in the affinity of manganese for the protein between pH 6.0 and 8.5 that seems to involve deprotonation of an acido-basic couple. Taken together, these results suggest that flexibility of critical regions of the protein, ionization of the cysteine 99 residue and improved K(D) values for the catalytic metal ion are coupled events. We propose that the molecular events observed in the isolated protein may be required for DivK activation and that they may be achieved in vivo through the specific protein-protein interactions between the response regulator and its cognate kinases.