Synthesis and structure–activity relationships of 2- and/or 4-halogenated 13β- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis

Ring A halogenated 13α-, 13β-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17β-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure–activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17β-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.     

A long-range physical map of human Chromosome 21q22.1 band from the YAC continuum

The human Chromosome (Chr) 21q22.1 region contains several genes for cytokines and neurotransmitters and the gene for superoxide dismutase (mutant forms of which can cause familial amyotrophic lateral sclerosis). A region of approximately 5.8 Mb encompassing D21S82 and the glycinamide ribonucleotide transformylase (GART) loci was covered by overlapping YAC clones, which were contiguously ordered by clone walking with sequence-tagged site (STSs). A total of 76 markers, including 29 YAC end-specific STSs, were unambiguously ordered in this 5.8-Mb region, and the average interval between markers was 76 kb. Restriction maps of the YAC clones with rare-cutting enzymes were simultaneously prepared, and the restriction sites were aligned to obtain a consensus restriction map of the proximal region of the 21q22.1 band. The restriction map made from 44 overlapping YACs contains 54 physically assigned STSs. By integrating the consensus map of the adjacent 1.8-Mb region, we obtained a fine physical map spanning 6.5 Mb of human Chr 21q22.1. This map contains 24 precisely positioned end-specific STSs and 12 NotI-linking markers. More than 39 potential CpG islands were identified in this region and were found to be unevenly distributed. This physical map and the YACs should be useful as a reference map and as a resource for further structural analysis of the Giemsa-negative band (R-band) of Chr 21q22.1. Received: 1 September 1995 / Accepted: 21 November 1995     

Mapping of the gene family for human heat-shock protein 90 alpha to chromosomes 1, 4, 11, and 14.

The HSP90 family of heat-shock proteins (encoded by genes for HSP90 alpha and beta) constitutes one of the major groups of proteins that are synthesized at increased rates in response to heat and other forms of stress. We previously isolated two distinct cDNA clones for HSP90 alpha from human peripheral blood lymphocytes and from HeLa cells transfected with the adenovirus E1A gene, respectively. To determine the organization of this complex multigene family in the human genome, we used three complementary approaches: Southern analysis of a panel of human/hamster somatic cell hybrids, molecular cloning of the cosmid HSP90 alpha clones from libraries prepared with DNAs from human lymphoblastoid cells, and in situ hybridization to human chromosomes. We demonstrate here that nucleotide sequences that encode HSP90 alpha map to human chromosomes 1q21.2-q22, 4q35, 11p14.1-p14.2, and 14q32.3. The chromosomal mapping of the loci, HSPCAL1, HSPCAL2, HSPCAL3, HSPCAL4, and the characterization of the respective genes should facilitate clarification of the organization of this gene family and lead to a better understanding of the biological functions of the gene product.     

Discovery of pyrrolopyridazines as novel DGAT1 inhibitors

A new structural class of DGAT1 inhibitors was discovered and the structure–activity relationship was explored. The pyrrolotriazine core of the original lead molecule was changed to a pyrrolopyridazine core providing an increase in potency. Further exploration resulted in optimization of the propyl group at C7 and the discovery that the ester at C6 could be replaced by five-membered heterocyclic rings. The analogs prepared have DGAT1 IC₅₀ values ranging from >10 μM to 48 nM.     

Genome sequencing of the NIES Cyanobacteria collection with a focus on the heterocyst-forming clade

Cyanobacteria are a diverse group of Gram-negative prokaryotes that perform oxygenic photosynthesis. Cyanobacteria have been used for research on photosynthesis and have attracted attention as a platform for biomaterial/biofuel production. Cyanobacteria are also present in almost all habitats on Earth and have extensive impacts on global ecosystems. Given their biological, economical, and ecological importance, the number of high-quality genome sequences for Cyanobacteria strains is limited. Here, we performed genome sequencing of Cyanobacteria strains in the National Institute for Environmental Studies microbial culture collection in Japan. We sequenced 28 strains that can form a heterocyst, a morphologically distinct cell that is specialized for fixing nitrogen, and 3 non-heterocystous strains. Using Illumina sequencing of paired-end and mate-pair libraries with in silico finishing, we constructed highly contiguous assemblies. We determined the phylogenetic relationship of the sequenced genome assemblies and found potential difficulties in the classification of certain heterocystous clades based on morphological observation. We also revealed a bias on the sequenced strains by the phylogenetic analysis of the 16S rRNA gene including unsequenced strains. Genome sequencing of Cyanobacteria strains deposited in worldwide culture collections will contribute to understanding the enormous genetic and phenotypic diversity within the phylum Cyanobacteria.     

The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1β (IL-1β), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

This study shows that inflammatory responses of microglial cells are markedly influenced by the ion transporter, NKCC1; blockade or genetic deletion of microglial NKCC1 has broad cell-autonomous effects, leading to changes in morphology, membrane conductance, process recruitment after injury, and cytokine production, with worsened neurological outcome after stroke.     

Mouse DNA primase plays the principal role in determination of permissiveness for polyomavirus DNA replication.

We have investigated the species-specific replication of polyomavirus DNA in the cell-free system that was established previously (Y. Murakami, T. Eki, M. Yamada, C. Prives, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 83:6347-6351, 1986). Extracts from various species of cells supported polyomavirus DNA replication in a species-specific manner that was consistent with the host range specificity of polyomavirus; extracts prepared from mouse and hamster cells were active, whereas extracts prepared from human, monkey, and insect cells were inactive. The addition of DNA polymerase alpha-primase purified from mouse cells induced the replication of polyomavirus DNA in a cell-free system containing polyomavirus large tumor antigen and nonpermissive cell extracts, such as human and insect cell extracts. Isolated mouse DNA primase alone also induced polyomavirus DNA replication in human cell extracts but not in insect cell extracts, indicating that mouse DNA primase plays the principal role in determining permissiveness for polyomavirus DNA replication.     

Development and characterization of polyclonal antibodies against the aryl hydrocarbon receptor protein family (AHR1, AHR2, and AHR repressor) of Atlantic killifish Fundulus heteroclitus

The aryl hydrocarbon receptor (AHR) and AHR repressor (AHRR) proteins regulate gene expression in response to some halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. The Atlantic killifish is a valuable model of the AHR signaling pathway, but antibodies are not available to fully characterize AHR and AHRR proteins. Using bacterially expressed AHRs, we developed specific and sensitive polyclonal antisera against the killifish AHR1, AHR2, and AHRR. In immunoblots, these antibodies recognized full-length killifish AHR and AHRR proteins synthesized in rabbit reticulocyte lysate, proteins expressed in mammalian cells transfected with killifish AHR and AHRR constructs, and AHR proteins in cytosol preparations from killifish tissues. Killifish AHR1 and AHR2 proteins were detected in brain, gill, kidney, heart, liver, and spleen. Antisera specifically precipitated their respective target proteins in immunoprecipitation experiments with in vitro-expressed proteins. Killifish ARNT2 co-precipitated with AHR1 and AHR2. These sensitive, specific, and versatile antibodies will be valuable to researchers investigating AHR signaling and other physiological processes involving AHR and AHRR proteins.