Protease-activated receptor-1 (hPar1), a survival factor eliciting tumor progression

Although ample evidence point to the central involvement of protease activated receptor-1 (PAR1) in tumor progression, little is known about the fate of the tumor when hPar1 is being silenced. We observed that hPar1 antisense clones exhibit low PAR1 levels, attenuated cell proliferation and invasion in vitro, and tumor formation in vivo. These clones showed noticeably reduced paxillin phosphorylation compared with the parental A375SM cells, whereas no change in the integrin levels was noticed. Antisense clones injected into the mice resulted in very few and only occasional small tumors, whereas advanced and vascularized tumors were observed in A375SM cells. The antisense-derived tumor sections expressed active caspase-3, increased terminal deoxynucleotidyl transferase-mediated nick-end labeling staining, and a markedly reduced proliferating cell nuclear antigen level compared with A375SM cell-derived tissue sections. Likewise, ablation of the hPar1 gene in a tetracycline-inducible hPar1 system leads to apoptosis in immature blood vessels, whereas mature vessels were unaffected. The activation of PAR1-induced pAkt/protein kinase B abrogated serum-deprived Bim(EL) induction and also markedly inhibited Bax levels. On the other hand, small interfering RNA silencing of the hPar1 gene induced the expression of Bim(EL), a direct substrate of Akt/protein kinase B and also induced expression of active caspase-9 and caspase-3. These results altogether identify PAR1 as a survival factor that protects cells from undergoing apoptosis. We conclude that whereas PAR1 gene expression correlates with tumor progression, its neutralization effectively initiates an apoptotic pathway leading at least in part to significantly reduced tumor formation.     

The latency of the light response is modulated by the phosphorylation state of Drosophila TRP at a specific site

Drosophila photoreceptors respond to oscillating light of high frequency (～100 Hz), while increasing the oscillating light intensity raises the maximally detected frequency. Recently, we reported that dephosphorylation of the light-activated TRP ion channel at S936 is a fast, graded, light-, and Ca²⁺-dependent process. We further found that this process affects the detection limit of high frequency oscillating light. Accordingly, transgenic Drosophila, which do not undergo phosphorylation at the S936-TRP site (trp^S936A), revealed a short time-interval before following the high stimulus frequency (oscillation-lock response) in both dark- and light-adapted flies. In contrast, the trp^S936D transgenic flies, which mimic constant phosphorylation, showed a long-time interval to oscillation-lock response in both dark- and light-adapted flies. Here we extend these findings by showing that dark-adapted trp^S936A flies reveal light-induced current (LIC) with short latency relative to trp^WT or trp^S936D flies, indicating that the channels are a limiting factor of response kinetics. The results indicate that properties of the light-activated channels together with the dynamic light-dependent process of TRP phosphorylation at the S936 site determine response kinetics.     

Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo

Like alternative splicing, leaky ribosomal scanning (LRS), which occurs at suboptimal translational initiation codons, increases the physiological flexibility of the genome by allowing alternative translation. Comprehensive analysis of 22208 human mRNAs indicates that, although the most important positions relative to the first nucleotide of the initiation codon, −3 and +4, are usually such that support initiation (A₋₃ = 42%, G₋₃ = 36% and G₊₄ = 47%), only 37.4% of the genes adhere to the purine (R)₋₃/G₊₄ rule at both positions simultaneously, suggesting that LRS may occur in some of the remaining (62.6%) genes. Moreover, 12.5% of the genes lack both R₋₃ and G₊₄, potentially leading to sLRS. Compared with 11 genes known to undergo LRS, 10 genes with experimental evidence for high fidelity A₊₁T₊₂G₊₃ initiation codons adhered much more strongly to the R₋₃/G₊₄ rule. Among the intron-less histone genes, only the H3 genes adhere to the R₋₃/G₊₄ rule, while the H1, H2A, H2B and H4 genes usually lack either R₋₃ or G₊₄. To address in vivo the significance of the previously described LRS of H4 mRNAs, which results in alternative translation of the osteogenic growth peptide, transgenic mice were engineered that ubiquitously and constitutively express a mutant H4 mRNA with an A₊₁→T₊₁ mutation. These transgenic mice, in particular the females, have a high bone mass phenotype, attributable to increased bone formation. These data suggest that many genes may fulfill cryptic functions by LRS.     

Evening chronotype and sleepiness predict impairment in executive abilities and academic performance of adolescents

The study aim was to better understand sleep and sleep-related factors affecting everyday executive capacities and academic performance among healthy adolescents. A cross-sectional survey on sleep, phase preference, academic performance and executive functions of high-school students was conducted. Female gender, grade status, sleepiness and evening chronotype accounted for approximately 25–30% of the variance in daily executive ability. Sleep duration was a weak predictor of executive skills. Lower school grades were associated with increased sleepiness, evening preference and poorer executive skills. These findings support the need for health education on ways to attenuate sleepiness and delayed phase in this population.     

Classroom Enters the Courtroom: Stereochemistry of SN1 and SN2 Reactions in Enantiomer Patent Litigations of the Antidepressant Escitalopram

The role of elementary stereochemistry is illustrated in the patent litigations of the blockbuster antidepressant drug escitalopram oxalate. An undergraduate student of organic chemistry would recognize the stereochemical courses of the intramolecular S_N2 and S_N1 reactions of the single‐enantiomer (S)‐diol intermediate in the synthesis of the blockbuster antidepressant drug escitalopram oxalate: retention of configuration of the chiral carbon atom under basic conditions and racemization under acidic conditions, respectively. He/she, in searching for a stereoselective ring‐closure reaction of the enantiomeric diol, will think of an S_N2 reaction in a basic medium. From these points of view, the process claim in the enantiomer patents of escitalopram is obvious/lacks an inventive step. An organic chemistry examination problem based on this scenario is offered. Chirality 28:39–43, 2016. © 2015 Wiley Periodicals, Inc.     

Post-proliferative cyclin E-associated kinase activity in differentiated osteoblasts: Inhibition by proliferating osteoblasts and osteosarcoma cells

Spontaneous differentiation of normal diploid osteoblasts in culture is accompanied by increased cyclin E associated kinase activity on (1) the retinoblastoma susceptibility protein pRB, (2) the p107 RB related protein, and (3) two endogenous cyclin E-associated substrates of 78 and 105 kD. Activity of the differentiation-related cyclin E complexes (diff.ECx) is not recovered in cdc2 or cdk2 immunoprecipitates. Phosphorylation of both the 105 kD endogenous substrate and the p107 exogenous substrate is sensitive to inhibitory activity (diff.ECx-i) present in proliferating osteoblasts. This inhibitory activity is readily recruited by the cyclin E complexes of differentiated osteoblasts but is not found in cyclin E immunoprecipitates of the proliferating cells themselves. Strong inhibitory activity on diff.ECx kinase activity is excerted by proliferating ROS 17/2.8 osteosarcoma cells. However, unlike the normal diploid cells, the diff.ECx-i activity of proliferating ROS 17/2.8 cells is recovered by cyclin E immunoprecipitation. The cyclin-dependent kinase inhibitor p21^CIP1/WAF1 inhibits diff.ECx kinase activity. Thus, our results suggest the existence of a unique regulatory system, possibly involving p21^CIP1/WAF1, in which inhibitory activity residing in proliferating cells is preferentially targeted towards differentiation-related cyclin E-associated kinase activity. J. Cell. Biochem. 66:141-152, 1997. © 1997 Wiley-Liss, Inc.