Identification of tammar wallaby SIRH12, derived from a marsupial-specific retrotransposition event

In humans and mice, there are 11 genes derived from sushi-ichi related retrotransposons, some of which are known to play essential roles in placental development. Interestingly, this family of retrotransposons was thought to exist only in eutherian mammals, indicating their significant contributions to the eutherian evolution, but at least one, PEG10, is conserved between marsupials and eutherians. Here we report a novel sushi-ichi retrotransposon-derived gene, SIRH12, in the tammar wallaby, an Australian marsupial species of the kangaroo family. SIRH12 encodes a protein highly homologous to the sushi-ichi retrotransposon Gag protein in the tammar wallaby, while SIRH12 in the South American short-tailed grey opossum is a pseudogene degenerated by accumulation of multiple nonsense mutations. This suggests that SIRH12 retrotransposition occurred only in the marsupial lineage but acquired and retained some as yet unidentified novel function, at least in the lineage of the tammar wallaby.     

Direct electrochemistry of engineered cytochrome b562 molecules with a ligand binding pocket

The rapid and reversible electron transfer reaction of cytochrome b562 was observed at an In2O3 electrode. The estimated heterogeneous electron transfer rate constant (k0') was k0' > or = 5.0 x 10(-3) cm s(-1) at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 x 10(-3) and 1.8 x 10(-3) cm s(-1), respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b562 was shifted negatively by approximately 135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.     

Discovery of diphenylcarbamate derivatives as highly potent and selective IP receptor agonists: orally active prostacyclin mimetics. Part 3

The new classes of diphenylcarbamate derivatives with a tetrahydronaphthalene skeleton as highly potent and selective IP agonists have been discovered. The optimized diphenylcarbamate type compound FK-788: (R)-4 exhibited potent antiaggregative potency with an IC50 of 18 nM and high binding affinity for the human recombinant IP receptor with K(i) values of 20 nM and selectivity for human IP over all other members of the human prostanoid receptor family. Compound (R)-4 was shown to exhibit good pharmacokinetic properties in rats and dogs, and also good bioavailability in healthy volunteers.     

Electrochemical evaluation of cellular physiological status under stress in Escherichia coli with the rpoS-lacZ reporter gene

We developed an electrochemical detection method for evaluating cellular physiological status based on the stringent response as a means to monitor cell viability. A reporter plasmid was constructed by inserting the beta-galactosidase gene (lacZ) under the control of the rpoS promoter, and then used to transform E. coli cells. Electrochemical responses from the products catalyzed by beta-galactosidase expressed by these E. coli cells were detected using the chronoamperometric technique in a nondestructive manner. Comparisons of response currents between the relA-positive strain and relA-negative strain revealed that increases in these currents were caused by the stringent response due to the stressful alcoholic environment, and thus as a model of stressful cultivating conditions. The current was proportional to the beta-galactosidase activity assayed by a conventional method that required the destruction of cells. The cellular physiological status, which depends on the stringent response as a viability marker, therefore, could then be evaluated online with a current using the rpoS-lacZ reporter gene in the relA-positive strain without pretreatment.     

Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions.     

X-ray diffraction from a left ventricular wall of rat heart

We studied x-ray diffraction from the left ventricular wall of an excised, perfused whole heart of a rat using x rays from the third-generation synchrotron radiation facility, SPring-8. With the beam at right angles to the long axis of the left ventricle, well-oriented, strong equatorial reflections were observed from the epicardium surface. The reflections became vertically split arcs when the beam passed through myocardium deeper in the wall, and rings were observed when the beam passed into the inner myocardium of the wall. These diffraction patterns were explained by employing a layered-spiral model of the arrangement of muscle fibers in the heart. In a quiescent heart with an expanded left ventricle, the muscle fibers at the epicardium surface were found to have a (1,0) lattice spacing smaller than in the rest of the wall. The intensity ratio of the (1,0) and (1,1) equatorial reflections decreased on contraction with a similar time course in all parts of the wall. The results show that it is possible to assign the origin of reflections in a diffraction diagram from a whole heart. This study offers a basis for interpretation of x-ray diffraction from a beating heart under physiologically and pathologically different conditions.     

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

Delivery of antibody-captured proteins into living cells using PTD-fused protein A

Protein transduction domain (PTD)-mediated protein delivery into animal cells is a useful technique for regulating cellular functions. Proteins captured by antibodies were delivered into living cells using an antibody/PTD-fused protein A complex. As a model protein, fluorescent-modified antibodies, captured by their respective primary antibody, were analyzed by fluorescence-activated cell sorting (FACS) which showed that the fluorescent-modified antibodies were directly delivered into cells. Peroxidase, captured by its specific antibody, was also delivered into cells and retained its activity.     

Geranylgeranyl-pyrophosphate (GGPP) synthase is down-regulated during differentiation of osteoblastic cell line MC3T3-E1

Isoprenylation of geranylgeranyl-pyrophosphate (GGPP) is critical for activation of small GTPases. We examined the roles of GGPP synthase (GGPPS) during the differentiation induced by the cell-to-cell contact in osteoblastic cell line MC3T3-E1 cells. We found that (1) both mRNA and protein expression of GGPPS was reduced with decrement of its activity during the differentiation, (2) GGOH, which is converted to GGPP in the cells, inhibited differentiation. These results suggest that the decrement of GGPP is critical for the cell-to-cell contact-induced differentiation, in which the down-regulation of GGPPS might be involved.     

Rhythmic expression of circadian clock genes in human leukocytes and beard hair follicle cells

Evaluating individual circadian rhythm traits is crucial for understanding the human biological clock system. The present study reports characterization of physiological and molecular parameters in 13 healthy male subjects under a constant routine condition, where interfering factors were kept to minimum. We measured hormonal secretion levels and examined temporal expression profiles of circadian clock genes in peripheral leukocytes and beard hair follicle cells. All 13 subjects had prominent daily rhythms in melatonin and cortisol secretion. Significant circadian rhythmicity was found for PER1 in 9 subjects, PER2 in 3 subjects, PER3 in all 13 subjects, and BMAL1 in 8 subjects in leukocytes. Additionally, significant circadian rhythmicity was found for PER1 in 5 of 8 subjects tested, PER2 in 2 subjects, PER3 in 6 subjects, and BMAL1 in 3 subjects in beard hair follicle cells. The phase of PER1 and PER3 rhythms in leukocytes correlated significantly with that of physiological rhythms. Our results demonstrate that leukocytes and beard hair follicle cells possess an endogenous circadian clock and suggest that PER1 and PER3 expression would be appropriate biomarkers and hair follicle cells could be a useful tissue source for the evaluation of biological clock traits in individuals.