Enantioresolution of fluorinated diphenylmethanols and determination of their absolute configurations by X-Ray crystallographic and 1H NMR anisotropy methods

Various fluorinated diphenylmethanols were enantioresolved by the methods of chiral camphorsultam-dichlorophthalic acid (CSDP acid) and/or 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid) yielding enantiopure alcohols. Their absolute configurations were unambiguously determined by X-ray crystallography of CSDP esters and/or by the (1)H NMR anisotropy method of MalphaNP esters for the first time.     

Effect of ethanol on cell growth of budding yeast: genes that are important for cell growth in the presence of ethanol

The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.     

Identification of cysteine protease inhibitors that belong to cystatin family 1 in the cellular slime mold Dictyostelium discoideum

Family 1 cystatins are cytosolic inhibitors of cysteine proteases, and they are conserved in higher eukaryotes. We characterized two newly identified family 1 cystatins of the cellular slime mold Dictyostelium discoideum, cystatin A1 and A2. Their recombinant proteins showed specific inhibitory activity against papain and cathepsin B, respectively. Using specific polyclonal antibodies, we found that cystatin A1 is stably expressed throughout the life cycle of Dictyostelium, whereas cystatin A2 expression is up-regulated during the course of development.     

Cloning and functional characterization of a new subtype of the amino acid transport system N

We have cloned a new subtype of theamino acid transport system N2 (SN2 or second subtype of system N) fromrat brain. Rat SN2 consists of 471 amino acids and belongs to therecently identified glutamine transporter gene family that consists ofsystem N and system A. Rat SN2 exhibits 63% identity with rat SN1. Italso shows considerable sequence identity (50-56%) with themembers of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung,stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediatesNa⁺-dependent transport of several neutral amino acids,including glycine, asparagine, alanine, serine, glutamine, andhistidine. The transport process is electrogenic, Li⁺tolerant, and pH sensitive. The transport mechanism involves the influxof Na⁺ and amino acids coupled to the efflux ofH⁺, resulting in intracellular alkalization. Proline,-(methylamino)isobutyric acid, and anionic and cationic amino acidsare not recognized by rat SN2.

     

Transient ischemia enhances tyrosine phosphorylation and binding of the NMDA receptor to the Src homology 2 domain of phosphatidylinositol 3-kinase in the rat hippocampus

Tyrosine phosphorylation of the NMDA receptor has been implicated in the regulation of the receptor channel. We investigated the effects of transient (15 min) global ischemia on tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B, and the interaction of NR2 subunits with the SH2 domain of phosphatidylinositol 3-kinase (PI3-kinase) in vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Transient ischemia induced a marked increase in the tyrosine phosphorylation of NR2A in both regions. The tyrosine phosphorylation of NR2B in CA3/dentate gyrus after transient ischemia was sustained and greater than that in CA1. PI3-kinase p85 was co-precipitated with NR2B after transient global ischemia. The SH2 domain of the p85 subunit of PI3-kinase bound to NR2B, but not to NR2A. Binding to NR2B was increased following ischemia and the increase in binding in CA3/dentate gyrus (4.5-fold relative to sham) was greater than in CA1 (1.7-fold relative to sham) at 10 min of reperfusion. Prior incubation of proteins with an exogenous protein tyrosine phosphatase or with a phosphorylated peptide (pYAHM) prevented binding. The results suggest that sustained increases in tyrosine phosphorylation and increased interaction of NR2B with the SH2 domain of PI3-kinase may contribute to altered signal transduction in the CA3/dentate gyrus after transient ischemia.     

Coordinated regulation of M phase exit and S phase entry by the Cdc2 activity level in the early embryonic cell cycle

In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle transition from M to S phase, we examined dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period, using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The result demonstrated that MCM binding to and the initiation of DNA replication of nuclear chromatin occurred depending on the decrease of Cdc2 activity to critical levels. Similarly, we found that Cdc2 inhibitory phosphorylation and cyclin B degradation were turned on and off, respectively, depending on the decrease in Cdc2 activity. However, their sensitivity to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle.     

Loss of cell cohesion in breast cytology as a characteristic of neuroendocrine carcinoma

OBJECTIVE: To characterize a specific group of breast cancers displaying a scattered single cell pattern in cytology and correlate it with histologic and immunohistochemical findings. STUDY DESIGN: Of 135 consecutive malignant breast cytologic specimens, 12 cases were selected for their scattered single cell pattern on aspiration cytology. Immunohistochemical staining for neuroendocrine markers and prognostic parameters was performed on paraffin sections of corresponding primary breast carcinomas. RESULTS: In the smears of the 12 cases, highly cellular neoplastic cells with a single cell pattern were observed predominantly. The tumor cells had relatively wide, granular cytoplasm and a low to moderate nuclear/cytoplasmic ratio. Histologically, they were arranged mainly in relatively large, solid nests and occasionally contained a tubular pattern with small amounts of stromal tissue. Five of the 12 cases demonstrated neuroendocrine differentiation with a positive immunoreaction for chromogranin A and synaptophysin. Except for the small mean size of the tumors (P < .01), no significant differences were identified among the prognostic parameters, including a nodal status, estrogen receptor status, growth fraction by Ki-67 or immunoreactivity for c-erbB-2, as compared with the other 123 cases. CONCLUSION: Loss of cell cohesion in breast cytology is a good morphologic marker for identifying neuroendocrine breast carcinoma.