Expression of a novel secretory form (Crb1s) of mouse Crumbs homologue Crb1 in skin development

Drosophila Crumbs and the mammalian homologues encoded by the Crb genes are transmembrane proteins required for determination of retinal cell polarity. We cloned a novel variant of mouse Crb1 and termed it Crb1s. Since the 3'-end of exon 6 remained unspliced, Crb1s coded for a short secretory protein lacking the transmembrane and cytoplasmic domains required for the function of Crb1. The Crb1 expression was confined to brain and eye, whereas Crb1s was detectable in various tissues including skin, lung, and kidney in adult mice. Active expression of Crb1s, but not Crb1, was observed during the skin development, in which localization of the Crb1s protein was altered from the basal layer to the upper layers. Cultured mouse keratinocytes synthesized the Crb1s protein and secreted a 80 kDa processed form to the supernatant. After Ca(2+)-induced differentiation, Crb1s became associated with focal adhesions and cell-cell contacts. Crb1s may play a role distinct from that of Crb1 in epidermal tissue morphogenesis.     

Microbial communities associated with geological horizons in coastal subseafloor sediments from the sea of okhotsk

Microbial communities from a subseafloor sediment core from the southwestern Sea of Okhotsk were evaluated by performing both cultivation-dependent and cultivation-independent (molecular) analyses. The core, which extended 58.1 m below the seafloor, was composed of pelagic clays with several volcanic ash layers containing fine pumice grains. Direct cell counting and quantitative PCR analysis of archaeal and bacterial 16S rRNA gene fragments indicated that the bacterial populations in the ash layers were approximately 2 to 10 times larger than those in the clays. Partial sequences of 1,210 rRNA gene clones revealed that there were qualitative differences in the microbial communities from the two different types of layers. Two phylogenetically distinct archaeal assemblages in the Crenarchaeota, the miscellaneous crenarchaeotic group and the deep-sea archaeal group, were the most predominant archaeal 16S rRNA gene components in the ash layers and the pelagic clays, respectively. Clones of 16S rRNA gene sequences from members of the gamma subclass of the class Proteobacteria dominated the ash layers, whereas sequences from members of the candidate division OP9 and the green nonsulfur bacteria dominated the pelagic clay environments. Molecular (16S rRNA gene sequence) analysis of 181 isolated colonies revealed that there was regional proliferation of viable heterotrophic mesophiles in the volcanic ash layers, along with some gram-positive bacteria and actinobacteria. The porous ash layers, which ranged in age from tens of thousands of years to hundreds of thousands of years, thus appear to be discrete microbial habitats within the coastal subseafloor clay sediment, which are capable of harboring microbial communities that are very distinct from the communities in the more abundant pelagic clays.     

A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-compound degradation genes for seco-steroids and 3-ketosteroid dehydrogenase genes

In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Delta(1)-dehydrogenase and 3-ketosteroid-Delta(4)(5alpha)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9alpha-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.     

Nucleolin and the packaging signal, psi, promote the budding of human immunodeficiency virus type-1 (HIV-1)

Gag proteins of human immunodeficiency virus type 1 (HIV-1) play a pivotal role in the budding of the virion, in which the zinc finger motifs of the gag proteins recognize the packaging signal of genomic RNA. Nucleolin, an RNA-binding protein, is identified as a cellular protein that binds to murine leukemia virus (MuLV) gag proteins and regulates the viral budding, suggesting that HIV-1 gag proteins, the packaging signal, psi and nucleolin affect the budding of HIV-1. Here we report that nucleolin enhances the release of HIV-1 virions which contain psi. Furthermore, nucleolin and gag proteins form a complex incorporated into virions, and nucleolin promotes the infectivity of HIV-1. Our results suggest that an empty particle which contains neither nucleolin nor the genomic RNA is eliminated during the budding process, and this mechanism is beneficial for escape from the host immune response against HIV-1.     

Characterization of C1-metabolizing prokaryotic communities in methane seep habitats at the Kuroshima Knoll, southern Ryukyu Arc, by analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA genes

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (approximately 34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the delta-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 x 10(6) copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 x 10(6) copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in gamma-Proteobacteria and had a high copy number of mxaF (7.1 x 10(6) copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 x 10(2) copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C1 metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.     

A genetic link between Tbx1 and fibroblast growth factor signaling

Tbx1 haploinsufficiency causes aortic arch abnormalities in mice because of early growth and remodeling defects of the fourth pharyngeal arch arteries. The function of Tbx1 in the development of these arteries is probably cell non-autonomous, as the gene is not expressed in structural components of the artery but in the surrounding pharyngeal endoderm. We hypothesized that Tbx1 may trigger signals from the pharyngeal endoderm directed to the underlying mesenchyme. We show that the expression patterns of Fgf8 and Fgf10, which partially overlap with Tbx1 expression pattern, are altered in Tbx1(-/-) mutants. In particular, Fgf8 expression is abolished in the pharyngeal endoderm. To understand the significance of this finding for the pathogenesis of the mutant Tbx1 phenotype, we crossed Tbx1 and Fgf8 mutants. Double heterozygous Tbx1(+/-);Fgf8(+/-) mutants present with a significantly higher penetrance of aortic arch artery defects than do Tbx1(+/-);Fgf8(+/+) mutants, while Tbx1(+/+);Fgf8(+/-) animals are normal. We found that Fgf8 mutation increases the severity of the primary defect caused by Tbx1 haploinsufficiency, i.e. early hypoplasia of the fourth pharyngeal arch arteries, consistent with the time and location of the shared expression domain of the two genes. Hence, Tbx1 and Fgf8 interact genetically in the development of the aortic arch. Our data provide the first evidence of a genetic link between Tbx1 and FGF signaling, and the first example of a modifier of the Tbx1 haploinsufficiency phenotype. We speculate that the FGF8 locus might affect the penetrance of cardiovascular defects in individuals with chromosome 22q11 deletions involving TBX1.     

Cell density inversely regulates D- and L-aspartate levels in rat pheochromocytoma MPT1 cells

In a previous report (FEBS Lett. 434 (1998) 231), we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in rat pheochromocytoma 12 (PC12) cells. This unique amino acid is believed to act as a novel messenger in mammalian cell regulation. However, the dynamics of D-Asp homeostasis in mammalian cells is yet to be elucidated. In this communication, we demonstrate that D-Asp is also synthesized in MPT1 cells (a subclone of PC12 cells) and that the D- and L-Asp levels in cells are regulated by cell density of the culture. Our data show that D-Asp levels increase, while in contrast, L-Asp levels decrease as a function of increased cell density. Conversely, in PC12 cells, which do not express the glutamate transporter involved in the incorporation of D- and L-Asp into cells, L-Asp levels decrease upon cell density increase while D-Asp concentrations remain almost unchanged. The results indicate that the biochemical behaviors of D- and L-Asp in mammalian cells are distinct and that the cellular levels of these stereoisomers appear to be under different control mechanisms.     

Immunolocalization of CXC chemokine and recruitment of polymorphonuclear leukocytes in the rat molar periodontal tissue after topical application of lipopolysaccharide

This study investigated the recruitment of polymorphonuclear leukocytes (PMNs) and the immunolocalization of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-2 (CINC-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS; 5 mg/ml) from Escherichia coli into the rat molar gingival sulcus. In normal periodontal tissues, a small number of MIP-2- and CINC-2-positive cells were seen in junctional epithelium (JE), especially in its coronal half. After topical application of LPS, a prominent increase of MIP-2- and CINC-2-positive JE cells was observed. Almost all JE cells strongly expressed them at day 1 and day 2, and then the number of chemokine-positive cells returned to normal at day 7. Corresponding to these chemokine expressions, LPS application induced a significant increase in the number of PMNs in the sub-JE area from 1 h to 2 days and a significant increase in JE area from 3 h to 5 days, indicating a dynamic flow of PMNs from the sub-JE area into JE. These findings indicated that JE cells produced MIP-2 and CINC-2 in response to LPS stimulation and suggested that MIP-2 and CINC-2 may be responsible for PMN migration toward the periodontal pathogen and may play an important role in the initiation of inflammation and subsequent periodontal tissue destruction.     

Synthesis and biological activity of a novel squalene epoxidase inhibitor, FR194738

The synthesis and biological properties of a novel squalene epoxidase inhibitor, FR194738, are described. This compound displayed potent in vitro inhibitory activities against squalene epoxidase and cholesterol synthesis, and lowered plasma cholesterol and triglyceride levels in dogs.