A novel homologue of the TIAP/m-survivin gene

The inhibitor of apoptosis (IAP) proteins comprise a highly conserved gene family that prevents cell death in response to a variety of stimuli. TIAP/m-survivin, a murine homologue of human Survivin, is a member of the IAP family. TIAP/m-survivin has one baculovirus IAP repeat (BIR) and lacks a C-terminal RING finger motif. Here we identified the genomic DNA region (TIAP-2) that is homologous to the TIAP/m-survivin gene by a low stringency genomic DNA hybridization. The region is on the chromomsome 9 which is distinct from that (chromosome 11) of the TIAP/m-survivin gene, and contains DNA sequence similar to a part of the BIR and the 3' side of the TIAP/m-survivin gene and the sequence homology between them is 92%. Expression of TIAP-2 mRNA was detected in various murine tissues by RT-PCR. Although expression of TIAP/m-survivin mRNA is upregulated in synchronized cells at S to G2/M phase of the cell cycle, expression of TIAP-2 mRNA was constant in the cell cycle, suggesting the different role of TIAP-2 from that of TIAP/m-survivin.     

Comparison of ES cell fate in sandwiched aggregates and co-cultured aggregates during blastocyst formation by monitored GFP expression

Markers and the means to detect them are required to monitor the fate of living cells. However, few suitable markers for living cells were known until a green fluorescent protein (GFP) was discovered. We have established mouse embryonic stem (ES) cell lines that express mutant GFP under the chicken beta-actin (CAG) promoter. Using these cell lines, we were able to follow the migration of ES cells during blastocyst formation both in sandwiching and coculture methods, even if only a single ES cell was used. Furthermore, the contribution of ES cells to the inner cell mass (ICM) was easily estimated at the blastocyst stage. We compared sandwiching with coculture aggregation relative to the contribution of the ES cell in the ICM, and the results indicated that there was no difference in the ratios of chimeric embryos having ICM contributed from cultured ES cells. Furthermore, an aggregated single ES cell was able to contribute three or four cells to the ICM at the blastocyst stage. Thus we conclude that one, instead of two, embryos is enough to make aggregation with ES cells, and a single ES cell attached to an embryo is enough to produce germline chimeras. Moreover, we could clearly observe single cell fate during blastocyst formation. This suggests that our established cell line can be used for monitoring single cell fate in vivo. In addition, we have shown that up to five doses of 30 sec of UV irradiation using GFP filters have no effect on the embryonic development.     

Why do chlorosomal chlorophylls lack the C132 -methoxycarbonyl moiety? An in vitro model study

Effects of the C13²-methoxycarbonyl moiety on the self-assembly of chlorosomal chlorophylls (Chls) were studied. Model compounds, zinc methyl 3-devinyl-3-(1-hydroxymethyl)-pheophorbides a and a (Zn-3¹-OH-Chls a/a, C13²-epimers) were synthesized from Chl a, and their aggregation behaviors were examined in Triton X-100 (TX-100) micellar suspensions and in 6%THF/water, in comparison with those of a pyrolized derivative, zinc methyl 3-devinyl-3-(1-hydroxymethyl)-13²-demethoxycarbonyl-pheophorbide a (Zn-3¹-OH-pyroChl a). Zn-3¹-OH-Chl a formed self-aggregates in the TX-100 micellar suspension and gave a Q_y absorption peak at 703 nm, while Zn-3¹-OH-pyroChl a aggregates of a Q_y peak at 740 nm. In the Zn-3¹-OH-Chl a aggregate spectrum, the Q_y red-shift was smaller, the band shape was broader, and the contribution of the residual monomer was more intense than that in the Zn-3¹-OH-pyroChl a aggregate spectrum. The bulky C13²-moiety limits the ways of molecular association, and electronic interaction between the component molecules of the Zn-3¹-OH-Chl a aggregate is weakened. Stereoselective control of the aggregation of the C13²-epimer was also examined.     

Structural and biochemical analyses of hemimethylated DNA binding by the SeqA protein

The Escherichia coli SeqA protein recognizes the 11 hemimethylated G-^mA-T-C sites in the oriC region of the chromosome, and prevents replication over-initiation within one cell cycle. The crystal structure of the SeqA C-terminal domain with hemimethylated DNA revealed the N⁶-methyladenine recognition mechanism; however, the mechanism of discrimination between the hemimethylated and fully methylated states has remained elusive. In the present study, we performed mutational analyses of hemimethylated G-^mA-T-C sequences with the minimal DNA-binding domain of SeqA (SeqA_71–181), and found that SeqA_71–181 specifically binds to hemimethylated DNA containing a sequence with a mismatched ^mA:G base pair [G-^mA(:G)-T-C] as efficiently as the normal hemimethylated G-^mA(:T)-T-C sequence. We determined the crystal structures of SeqA_71–181 complexed with the mismatched and normal hemimethylated DNAs at 2.5 and 3.0 Å resolutions, respectively, and found that the mismatched ^mA:G base pair and the normal ^mA:T base pair are recognized by SeqA in a similar manner. Furthermore, in both crystal structures, an electron density is present near the unmethylated adenine, which is only methylated in the fully methylated state. This electron density, which may be due to a water molecule or a metal ion, can exist in the hemimethylated state, but not in the fully methylated state, because of steric clash with the additional methyl group.     

Imaging analysis of subcellular correlation of androgen receptor and estrogen receptor alpha in single living cells using green fluorescent protein color variants

Androgen and estrogen act not only in a sex-specific manner but also interactively and synergistically. In the present study, to examine the possible interaction between androgen receptor (AR) and estrogen receptor-alpha (ERalpha), we investigated the subcellular dynamics of AR and ERalpha fused with green fluorescent protein color variants in single living cells using time-lapse microscopy and the technique of fluorescence recovery after photobleaching. AR and ERalpha showed punctate colocalization in the nucleus with estrogen, but not androgen. N-terminal AR deletion mutant did not form a nuclear punctate pattern with either androgen or estrogen. In the presence of AR, but not ERalpha, N-terminal AR deletion mutant formed a punctate nuclear pattern with androgen. AR had different mobility depending on the ligand and the presence of ERalpha. On the other hand, AR had little effect on the stability of ERalpha. ERalpha mutant that does not bind coactivators did not alter the mobility of AR. Taken together, using an imaging technique, we clarified that possible homo/hetero dimerization between AR and ERalpha could be attributed to androgen-estrogen interaction in living cells.     

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole induces apoptosis and necrosis with activation of different caspases in rat splenocytes

A dietary carcinogen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) at 20 microM activates caspase-3-like proteases as an apoptotic marker in rat splenocytes. The present study demonstrated 100 microM Trp-P-1 induced necrosis with activation of caspase-3-like proteases. The activation in necrosis and apoptosis resulted from the activation of caspase-9 and caspase-8, respectively. Thus, Trp-P-1 induces apoptosis and necrosis with the activation of different caspases.     

Enhanced calcium absorption in the small intestine by a phytate-removed deamidated soybean globulin preparation

Soybean globulins were deamidated after removing phytate using ion-exchange resins, and then hydrolyzed by digestive enzymes. The phytate-removed deamidated soybean globulins (PrDS) retained high calcium-binding ability even after the hydrolysis by digestive enzymes. PrDS and its hydrolysates enhanced calcium absorption from the small intestine when injected into the small intestine together with a calcium solution.