Combination therapy with transductive anti-death FNK protein and FK506 ameliorates brain damage with focal transient ischemia in rat

Many practical therapies have been explored as clinical applications for ischemic cerebral infarction; however, most are still insufficient to treat acute stroke. We show here a potential combination therapy in a rat focal ischemic model to improve neurological symptoms as well as to reduce infarct volumes at the maximum level. We applied protein transduction technology using artificial anti-death Bcl- x _l derivative with three amino acid-substitutions (Y22 F , Q26 N and R165 K ) (FNK) protein fused with a protein-transduction-domain peptide (PTD-FNK). When PTD-FNK was administrated 1 h after initiating ischemia followed by the administration of an immunosuppressant FK506 with a 30-min time lag, infarct volumes of the total brain and cortex were markedly reduced to 27% and 14%, respectively. This procedure not only reduced the infarct volume and edema, but also markedly improved neurological symptoms. The therapeutic effect continued for at least 1 week after ischemia. FK506 inhibited the transduction of PTD-FNK in vitro , which explains the requirement of a time lag for the administration of FK506. An additional in vitro experiment showed that PTD-FNK, when administered 30 min before FK506, gave the maximal protective effect by reducing the intracellular calcium concentration. We propose that this combination therapy would provide a synergistic protective effect by both drugs, reducing adverse the effects of FK506.     

Effects of redecoration of a hospital isolation room with natural materials on stress levels of denizens in cold season

We investigated the effects of redecoration of a hospital isolation room with natural materials on thermoregulatory, cardiovascular and hormonal parameters of healthy subjects staying in the room. Two isolation rooms with almost bilaterally-symmetrical arrangements were used. One room (RD) was redecorated with wood paneling and Japanese paper, while the other (CN) was unchanged (with concrete walls). Seven healthy male subjects stayed in each room for over 24 h in the cold season. Their rectal temperature (T_re) and heart rate, and the room temperature (T_a) and relative humidity were continuously measured. Arterial blood pressures, arterial vascular compliance, thermal sensation and thermal comfort were measured every 4 h except during sleeping. Blood was sampled after the stay in the rooms. In RD, T_a was significantly higher by about 0.4°C and relative humidity was lower by about 5% than in CN. Diurnal T_re levels of subjects in RD significantly differed from those in CN, i.e., T_res were significantly higher in RD than in CN especially in the evening. In RD, the subjects felt more thermally-comfortable than in CN. Redecoration had minimal effects on cardiovascular parameters. Plasma levels of catecholamines and antidiuretic hormone did not differ, while plasma cortisol level was significantly lower after staying in RD than in CN by nearly 20%. The results indicate that, in the cold season, redecoration with natural materials improves the thermal environment of the room and contributes to maintaining core temperature of denizens at preferable levels. It also seems that redecoration of room could attenuate stress levels of isolated subjects.     

Design and synthesis of an artificial ladder-shaped polyether that interacts with glycophorin A

Ladder-shaped polyether (LSP) compounds, such as brevetoxins and ciguatoxins, are thought to interact with transmembrane (TM) proteins. As a model LSP compound, we designed and synthesized an artificial tetracyclic ether (1) and evaluated its interaction with glycophorin A (GpA), a membrane protein known to dimerize or oligomerize between membrane-integral -helical domains. Model compound 1 was found to induce the dissociation of oligomeric GpA in a similar manner to natural LSPs when examined by SDS–PAGE. The results suggest that even an artificial tetracyclic ether possesses the ability to interact with TM proteins, presumably through the intermolecular hydrogen bonds (C–H  O) with the GXXXG motif.     

Statistical Validation of Two Sample Comparison Methods for Oligonucleotide Microarray in Rat Ischemia Model

In gene expression analyses using a high-density oligonucleotide array in a rat ischemia model, two comparison methods, “pair-wise comparison” and “sample average comparison”, were evaluated based on statistical methods. The reliability of the elements screened with a 1.2 to 10-fold threshold was also evaluated. In pair-wise comparisons, most of the elements were significantly independent of the threshold value, with the percentage of significant elements remaining above 95%, when screened at 2.5-fold or higher threshold value. Pair-wise comparison structurally provided strict screening, which resulted in genes that were not selected despite significant alterations in expression. Screening by “sample average comparison” resulted in elements with low probability of significance, which suggested the necessity for increasing the reliability by additional statistical analyses after screening. When genes with altered expression were screened using an oligonucleotide array, marked differences in the numbers and reliability were proved to exist among elements screened by each sample comparison method.     

Negative regulation of Jak2 by its auto-phosphorylation at tyrosine 913 via the Epo signaling pathway

Janus kinase 2 (Jak2) has a pivotal role in erythropoietin (Epo) signaling pathway, including erythrocyte differentiation and Stat5 activation. In the course of screening for critical phosphorylation of tyrosine residues in Jak2, we identified tyrosine 913 (Y(913)) as a novel and functional phosphorylation site, which negatively regulates Jak2. Phosphorylation at Y(913) rapidly occurred and was sustained for at least 120 min after Epo stimulation, in contrast to the transient phosphorylation of Y(1007/1008) in the activation loop of Jak2. Interestingly, phosphorylation defective mutation of Y(913) (Y(913)F) results in a significant enhancement of Epo-induced Jak2 activation, whereas phosphorylation mimic mutation of Y(913) (Y(913)E) completely abrogated its activation. Furthermore, Jak2 deficient fetal liver cells expressing Y(913)F mutant generated many mature erythroid BFU-E and CFU-E colonies, while Y(913)E mutant failed to reconstitute Jak2 deficiency. We also demonstrate, in Jak1, phosphorylation of Y(939), a corresponding tyrosine residue with Y(913), negatively regulated Jak1 signaling pathway. Accordingly, our results suggest that this tyrosine phosphorylation in JH1 domain may be involved in common negative regulation mechanism for Jak family.     

Binding affinity of aluminium to human serum transferrin and effects of carbohydrate chain modification as studied by HPLC/high-resolution ICP-MS--speciation of aluminium in human serum

Aluminium (Al) in the blood is bound to transferrin (Tf), a glycoprotein of about 80kDa that is characterized by its need for a synergistic anion. In this focused review, the binding affinity of Al to Tf is surveyed in the context of our recent studies using on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry (HPLC/HR-ICP-MS). Al in human serum without any in vitro Al-spikes was present in a form bound to the N-lobe site of Tf. The influences of sialic acid in the carbohydrate chain of human serum Tf (hTf) were studied using asialo-hTf, obtained by treatment with sialidase. The binding affinity of Fe was similar between asialo-hTf and native-hTf, while that of Al for asialo-hTf was larger than that for native-hTf, especially in the presence of oxalate, a synergistic anion. The above findings are discussed in relation to diseases in which the serum concentrations of carbohydrate-deficient Tf and oxalate are augmented.