Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDnas identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse KIAA-homologous genes since 2001. As an extension of this project, we also started to accumulate mouse cDNA clones homologous to the human FLJ cDNA clones which are another long cDNA resource produced in our institute. We have isolated the cDNA clones from size-fractionated cDNA libraries derived from five different mouse tissues and natural killer T-cells. Although the human FLJ cDNA clones were originally derived from human spleen libraries, one-third of their mouse homologues were obtained from the brain library. We designated these homologues "mFLJ" plus a 5-digit number and herein characterized 110 mFLJ cDNA clones. We assigned an integrity of the CDSs from the comparison of the 110 cDNA clones with the corresponding human FLJ cDNA clones. The average size of the 110 mouse cDNA sequences was 3.8 kb and that of the deduced amino acid sequences from their longest CDS in each cDNA was 663 amino acid residues. Homology and/or motif search against public databases revealed new domains and/or motifs in 26 mFLJ gene products which provide additional speculation regarding the function of FLJ genes.     

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.     

Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice

The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation.     

Requirement for Tec kinases in chemokine-induced migration and activation of Cdc42 and Rac

Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. We demonstrate here that the Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induced transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk(-/-)Itk(-/-) mice exhibited defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. Expression of a dominant-negative Itk impaired SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells.     

An inverse correlation between expression of a preprocathepsin B-related protein with cysteine-rich sequences and steroid 11beta -hydroxylase in adrenocortical cells

A cDNA encoding a secretory protein hitherto unknown was cloned from mouse adrenocortical cells by subtractive hybridization between the cells without and with expressing steroid 11beta-hydroxylase (Cyp11b-1), a marker for the functional differentiation of cells in the zonae fasciculata reticularis (zFR). The deduced protein consisting of 466 amino acids contained a secretory signal, epidermal growth factor-like repeats, and a proteolytically inactive cathepsin B-related sequence. The amino acid sequence was 89% identical with that of human tubulointerstitial nephritis antigen-related protein. Among the mouse organs examined, adrenal glands prominently expressed its mRNA. The mRNA and its encoded protein were detected in the outer adrenocortical zones that do not express Cyp11b-1, i.e. the zona glomerulosa and the undifferentiated cell zone, while being undetectable in zFR that express Cyp11b-1. The new protein was designated as adrenocortical zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1 expression were established from Y-1 adrenocortical cells that originally express Cyp11b-1 but little AZ-1. Analyses of the clonal lines revealed that Cyp11b-1 is detected in the clonal lines maintaining little AZ-1 expression and becomes undetectable in those expressing AZ-1. On the other hand, irrespective of the AZ-1 expression, all clones expressed cholesterol side-chain cleavage enzyme, which occurs throughout the cortical zones. These results demonstrated that adrenocortical cells expressing AZ-1 do not express Cyp11b-1, whereas those with little AZ-1 express this zFR marker in vitro and in vivo, implying a putative role of AZ-1 in determining the zonal differentiation of adrenocortical cells.     

Triphenyltin enhances the neutrophilic differentiation of promyelocytic HL-60 cells

Triphenyltin (TPT) is an environmental endocrine disruptor and toxic substance, but little information is available on its immunological effects. To assess the effect of TPT on leukocyte differentiation, we investigated its effect on the neutrophilic differentiation of HL-60 cells induced by dimethyl sulfoxide and granulocyte colony-stimulating factor (G-CSF) for 6 days. At a low concentration, 10(-7)M, TPT increased superoxide production by differentiated HL-60 cells stimulated with opsonized zymosan (OZ) by about 45% and increased expression of CD18, a component of the OZ-receptor, by about 90%. Real-time PCR analysis revealed that TPT augmented the expression not only of CD18 but also of components of superoxide-generating NADPH-oxidase, p47phox, 2.7-fold, and p67phox, 2.0-fold, and of granulocyte colony-stimulating factor receptor (G-CSFR), 3.0-fold, whereas various other endocrine disruptors, including parathion, vinclozolin, and bisphenol A, had no such enhancing effects. The results of a DNA macroarray analysis showed that TPT enhanced the expression of G-CSFR and certain other neutrophil functional proteins, including CD14 and myeloid leukemia cell differentiation protein (MCL-1), and that TPT induced a decrease in expression of LC-PTP, leukocyte protein-tyrosine phosphatase, to about half the control level. The TPT-dependent suppression of LC-PTP was confirmed by real-time PCR analysis, and the results of immunoblotting indicated that TPT enhances the expression of myeloid specific tyrosine kinase hck by about 30% at the protein level, and this together with the reduction of LC-PTP may enhance tyrosine phosphorylation, in turn resulting in enhancement of superoxide production. These findings suggest that TPT may have an enhancing effect on the neutrophilic maturation of leukocytes.     

Dictyostelium myosin II mutations that uncouple the converter swing and ATP hydrolysis cycle

During the ATP hydrolysis cycle of the Dictyostelium myosin II motor domain, two conserved alpha-helices, the SH1/SH2 helix and the relay helix, rotate in a coordinated way to induce the swing motion of the converter domain. A network of hydrophobic and ionic interactions in these two helices and the converter may ensure that the motions of these helices are effectively transmitted to the converter. To examine the roles of these interactions in the ATPase-dependent converter swing, we disrupted two conserved hydrophobic linkages among them by means of a point mutation (I499A or F692A). The resulting mutations induced only limited changes in the kinetic parameters of ATP hydrolysis, except for a marked increase of basal MgATPase activity. However, the mutant myosins completely lost their in vitro and in vivo motor functions. Measurements of the intrinsic tryptophan fluorescence and the GFP-based FRET revealed that the converter domain of these mutants did not swing during steady-state ATP hydrolysis or in the presence of tightly trapped Mg.ADP.V(i), which shows that the point mutations induced the uncoupling of the converter swing and ATP hydrolysis cycle. These results highlight the importance of these hydrophobic linkages for transmitting the coordinated twist motions of the helices to the converter as well as the requirement of this converter swing for force generation.     

Antisense oligonucleotide to cofilin enhances respiratory burst and phagocytosis in opsonized zymosan-stimulated mouse macrophage J774.1 cells

Phagocytes play a central role in the host defense system, and the relationship between the mechanism of their activation and cytoskeletal reorganization has been studied. We have previously reported a possible involvement of cofilin, an actin-binding protein, in phagocyte functions through its phosphorylation/dephosphorylation and translocation to the plasma membrane regions. In this work, we have obtained a new line of evidence showing an important role of cofilin in phagocyte functions using the mouse macrophage cell line J774.1 and an antisense oligonucleotide to cofilin. Upon stimulation with opsonized zymosan (OZ), cofilin was phosphorylated, and it accumulated around phagocytic vesicles. As the antisense oligonucleotide to cofilin, a 20-mer S-oligo corresponding to the sequence including the AUG translational initiation site was found to be effective. In the cells treated with the antisense oligonucleotide, the amount of cofilin was less than 30% of that in the control cells, and the level of F-actin was two or three times higher than that in the control cells before and throughout the cell activation. In the antisense oligonucleotide-treated cells, OZ-triggered superoxide production was three times faster than that in the control cells. Furthermore, phagocytosis of OZ was enhanced by the antisense. These results show that cofilin plays an essential role in the control of phagocyte function through regulation of actin filament dynamics.