NIRF,a novel RING finger protein,is involved in cell-cycle regulation

Through database mining, we found a novel PEST-containing nuclear protein (PCNP). To characterize PCNP, we carried out yeast two-hybrid screening for PCNP-interacting factors. A novel Np95/ICBP90-like RING finger protein (NIRF), which possessed a ubiquitin-like domain, a PHD finger, a YDG/SRA domain and a RING finger, was identified. Interaction between PCNP and NIRF was clarified by mammalian two-hybrid system, GST pull-down assay, and nuclear co-localization. RT-PCR showed that NIRF expression is high in proliferating phase but significantly low in G0/G1 phase in normal TIG-7 and WI-38 cells, while consistently high in tumoral HT-1080 and HepG2 cells, suggesting that NIRF is involved in cell-cycle regulation. The NIRF gene resides in 9p23-24.1 that is altered in numerous types of tumors at the top of frequency. Furthermore, the NIRF gene is just within small amplicons in some tumors, suggesting that PCNP and NIRF might be involved in some aspects of tumorigenesis.     

Treatment of hypoglossia-hypodactyly syndrome without extremeity anomalies

Three cases of hypoglossia-hypodactyly syndrome without limb deformities are reported. All exhibited different degrees of tongue hypoplasia, micrognathia, retrognathia with a very narrow space between the left and right halves of the mandible, constricted isthmus, and only one lower incisor. Bone lengthening for the midline mandibular hypoplasia and orthodontic treatment were performed in the three cases with satisfactory results.     

Induction of apoptosis in mouse brain capillary endothelial cells by cyclosporin A and tacrolimus

Although cyclosporin A and tacrolimus are used clinically as potent immunosuppressants, there have been reports of neurotoxicity and encephalopathy. A possible mechanism is that these drugs damage the blood-brain barrier (BBB), inducing dysfunction and increased permeability, and are then able to enter the brain. We studied the cytotoxicity of cyclosporin A and tacrolimus, focused on apoptosis induction, using an immortalized cell line established from BALB/c mouse cerebral microvessel endothelial cells (MBEC4). We found that these two drugs induced cell shrinkage, chromatin condensation and DNA fragmentation, which are characteristics of apoptosis. Our data suggest that the induction of apoptosis on the brain capillary endothelial cells may be at least partly involved in the occurrence of immunosuppressant-induced encephalopathy.     

A Comprehensive,Multi-Scale Dynamical Model of ErbB Receptor Signal Transduction in Human Mammary Epithelial Cells

The non-receptor tyrosine kinase Src and receptor tyrosine kinase epidermal growth factor receptor (EGFR/ErbB1) have been established as collaborators in cellular signaling and their combined dysregulation plays key roles in human cancers, including breast cancer. In part due to the complexity of the biochemical network associated with the regulation of these proteins as well as their cellular functions, the role of Src in EGFR regulation remains unclear. Herein we present a new comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells. This model, constructed manually from published biochemical literature, consists of 245 nodes representing proteins and their post-translational modifications sites, and over 1,000 biochemical interactions. Using computer simulations of the model, we find it is able to reproduce a number of cellular phenomena. Furthermore, the model predicts that overexpression of Src results in increased endocytosis of EGFR in the absence/low amount of the epidermal growth factor (EGF). Our subsequent laboratory experiments also suggest increased internalization of EGFR upon Src overexpression under EGF-deprived conditions, further supporting this model-generated hypothesis.     

Characterization of rainbow trout branched-chain alpha-keto acid dehydrogenase complex: inter-domain segments of the E2 component affect the overall activity

Branched-chain alpha-keto acid dehydrogenase complex (BCKADH) contains decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3) as catalytic components. BCKADH purified from rainbow trout (Oncorhynchus mykiss) liver was comparable with mammalian BCKADH in various enzymatic characteristics, but less efficient in catalyzing the overall reaction. The trout E2 subunit was larger than the mammalian subunit and rather similar to the chicken one in relative molecular mass on SDS-PAGE, whereas the E1 component was similar between trout and mammalian both in relative molecular mass of its alpha and beta subunits and in the catalytic activity. Trout E2 cDNA cloning and nucleotide sequencing revealed that the mature trout E2 subunit consists of 435 residues, and possesses 14 additional residues compared with mammalian E2. Eleven of these are localized in two interdomain segments as two sequences with two and nine residues, respectively. Trout E2 was inferior to rat E2 in the capacity for binding the E1 component, similar to chicken E2. Thus, it appears that non-mammalian BCKADH E2 is distinct from that in mammals in the structure of interdomain segments, resulting in reduction of overall activity of the enzyme complex.     

A novel human nucleoside diphosphate (NDP) kinase,Nm23‐H6, localizes in mitochondria and affects cytokinesis

Nucleoside diphosphate kinases (NDP kinases) are enzymes known to be conserved throughout evolution and have been shown to be involved in various biological events, in addition to the “housekeeping” phosphotransferase activity. We present the molecular cloning of a novel human NDP kinase gene, termed Nm23‐H6. Nm23‐H6 gene has been mapped at chromosome 3p21.3 and is highly expressed in heart, placenta, skeletal muscle, and some of the cancer cell lines. Recombinant Nm23‐H6 protein has been identified to exhibit functional NDP kinase activity. Immunolocalization studies showed that both endogenous and inducibly expressed Nm23‐H6 proteins were present as short, filament‐like, perinuclear radical arrays and that they colocalized with mitochondria. Cell fractionation study also demonstrated the presence of Nm23‐H6 protein in a mitochondria‐rich fraction. Moreover, induction of overexpression of Nm23‐H6 in SAOS2 cells, using the Cre‐loxP gene activation system, resulted in growth suppression and generation of multinucleated cells. Flow cytometric analysis also demonstrated that the proportion of cells with more than 4N DNA content increased to 28.1% after induction of Nm23‐H6, coinciding with the appearance of multinucleated cells. These observations suggest that Nm23‐H6, a new member of the NDP kinase family, resides in mitochondria and plays a role in regulation of cell growth and cell cycle progression. J. Cell. Biochem. 76:254–269, 1999. © 1999 Wiley‐Liss, Inc.     

Biochemical and physiological changes induced by anthrax lethal toxin in J774 macrophage-like cells.

Experiments were performed to probe the mechanism by which Bacillus anthracis Lethal Toxin (LeTx) causes lysis of J774 macrophage-like cells. After incubation of cells with saturating concentrations of the toxin, two categories of effects were found, which were distinguishable on the basis of chronology, Ca(2+)-dependence, and sensitivity to osmolarity. The earliest events (category I), beginning 45 min postchallenge, were an increase in permeability to 22Na and 86Rb and a rapid conversion of ATP to ADP and AMP. Later events (category II) included alterations in membrane permeability to 45Ca, 51Cr, 36Cl, 35SO4, 3H-amino acids, and 3H-uridine, beginning at 60 min; inhibition of macromolecular synthesis, leakage of cellular lactate dehydrogenase and onset of gross morphological changes, at approximately 75 min; and cell lysis, beginning at 90 min. Category II events exhibited an absolute requirement for extracellular Ca2+ and were blocked by addition of 0.3 M sucrose to the medium, whereas category I events were attenuated, but not blocked, by either of these conditions. On the other hand, both ATP depletion and the category II events were blocked in osmotically stabilized medium that was also isoionic for Na+ and K+. This suggests that permeabilization of the plasma membrane to monovalent cations and water may be the earliest of the physiological changes described here. The resulting influx of Na+ and efflux of K+ would be expected to cause depletion of ATP, via increased activity of the Na+/K+ pump. Subsequently the influx of Ca2+, induced by depletion of ATP, imbalances in monovalent cautions, and/or more dramatic changes in permeability due to influx of water, would be expected to trigger widespread changes leading ultimately to cytolysis.