The molecular basis for genetic polymorphism of human deoxyribonuclease II (DNase II): a single nucleotide substitution in the promoter region of human DNase II changes the promoter activity

We report that, contrary to common belief, polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein are functionally displayed on the phage surface. In a phagemid display system, carboxy-terminal fusion through optimized linker sequences resulted in display levels comparable to those achieved with conventional amino-terminal fusions. These findings are of considerable importance to phage display technology because they enable investigations not suited to amino-terminal display, including the study of protein–protein interactions requiring free carboxy-termini, functional cDNA cloning efforts, and the display of intracellular proteins.     

Transient formation of ubisemiquinone radical and subsequent electron transfer process in the Escherichia coli cytochrome bo

To elucidate a unique mechanism for the quinol oxidation in the Escherichia coli cytochrome bo, we applied pulse radiolysis technique to the wild-type enzyme with or without a single bound ubiquinone-8 at the high-affinity quinone binding site (Q(H)), using N-methylnicotinamide (NMA) as an electron mediator. With the ubiquinone bound enzyme, the reduction of the oxidase occurred in two phases as judged from kinetic difference spectra. In the faster phase, the transient species with an absorption maximum at 440 nm, a characteristic of the formation of ubisemiquinone anion radical, appeared within 10 micros after pulse radiolysis. In the slower phase, a decrease of absorption at 440 nm was accompanied by an increase of absorption at 428 and 561 nm, characteristic of the reduced form. In contrast, with the bound ubiquinone-8-free wild-type enzyme, NMA radicals directly reduced hemes b and o, though the reduction yield was low. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone anion radical at the Q(H) site to heme b exists in cytochrome bo. The first-order rate constant of this process was calculated to be 1.5 x 10(3) s(-1) and is comparable to a turnover rate for ubiquinol-1. The rate constant for the intramolecular electron transfer decreased considerably with increasing pH, though the yields of the formation of ubisemiquinone anion radical and the subsequent reduction of the hemes were not affected. The pH profile was tightly linked to the stability of the bound ubisemiquinone in cytochrome bo [Ingledew, W. J., Ohnishi, T., and Salerno, J. C. (1995) Eur. J. Biochem. 227, 903-908], indicating that electron transfer from the bound ubisemiquinone at the Q(H) site to the hemes slows down at the alkaline pH where the bound ubisemiquinone can be stabilized. These findings are consistent with our previous proposal that the bound ubiquinone at the Q(H) site mediates electron transfer from the low-affinity quinol oxidation site in subunit II to low-spin heme b in subunit I.     

Mammalian deoxyribonucleases I are classified into three types: pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations

Deoxyribonuclease I (DNase I) activities were measured in 14 different tissues from humans and 5 other mammals (bovine, pig, rabbit, rat, and mouse) by using the single radial enzyme diffusion (SRED) method, which is a sensitive and nonradioactive assay for nucleases. The results indicated that these species are classifiable into three groups on the basis of their different tissue distributions of DNase I. In human and pig, the pancreas showed the highest activity of DNase I; in rat and mouse, the parotid glands showed the highest activity; and in bovine and rabbit, both pancreas and parotid glands showed high activity. Therefore we designated human and pig DNase I as pancreas type, rat and mouse DNase I as parotid type, and bovine and rabbit DNase I as pancreas-parotid (or mixed) type. DNase I of the pancreas type was more sensitive to low pH than the other types. DNase I of pancreas type is secreted into the intestinal tract under neutral pH conditions, whereas the other types are secreted from the parotid gland and have to pass through the very acidic conditions in the stomach. Differences in the tissue distribution and acid sensitivity of mammalian DNases I may provide important information about their digestive function from the evolutionary perspective.     

More than 95% reversal of left-right axis induced by right-sided hypodermic microinjection of activin into Xenopus neurula embryos

In recent years, genes that show left-right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula-neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus neurula embryos. After right-side injection of 10-250 pg activin protein, both early neurulae just after gastrulation movement (stage 13-14) and late neurulae just before neural tube closure (stage 17-18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1-50 pg) was microinjected into both sides of neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the neurula stage. Activin not only acts on the neurulae to cancel the original L-R specification up to the late neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of neurulae have equal potential for L-R differentiation.     

Resonance raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide

Cytochrome bo from Escherichia coli, a member of the heme-copper terminal oxidase superfamily, physiologically catalyzes reduction of O(2) by quinols and simultaneously translocates protons across the cytoplasmic membrane. The reaction of its ferric pulsed form with hydrogen peroxide was investigated with steady-state resonance Raman spectroscopy using a homemade microcirculating system. Three oxygen-isotope-sensitive Raman bands were observed at 805/X, 783/753, and (767)/730 cm(-)(1) for intermediates derived from H(2)(16)O(2)/H(2)(18)O(2). The experiments using H(2)(16)O(18)O yielded no new bands, indicating that all the bands arose from the Fe=O stretching (nu(Fe)(=)(O)) mode. Among them, the intensity of the 805/X cm(-)(1) pair increased at higher pH, and the species giving rise to this band seemed to correspond to the P intermediate of bovine cytochrome c oxidase (CcO) on the basis of the reported fact that the P intermediate of cytochrome bo appeared prior to the formation of the F species at higher pH. For this intermediate, a Raman band assignable to the C-O stretching mode of a tyrosyl radical was deduced at 1489 cm(-)(1) from difference spectra. This suggests that the P intermediate of cytochrome bo contains an Fe(IV)=O heme and a tyrosyl radical like compound I of prostaglandin H synthase. The 783/753 cm(-)(1) pair, which was dominant at neutral pH and close to the nu(Fe)(=)(O) frequency of the oxoferryl intermediate of CcO, presumably arises from the F intermediate. On the contrary, the (767)/730 cm(-)(1) species has no counterpart in CcO. Its presence may support the branched reaction scheme proposed previously for O(2) reduction by cytochrome bo.     

Phylogeography of the Coastal Mosquito Aedes togoi across Climatic Zones: Testing an Anthropogenic Dispersal Hypothesis

The coastal mosquito Aedes togoi occurs more or less continuously from subarctic to subtropic zones along the coasts of the Japanese islands and the East Asian mainland. It occurs also in tropical Southeast Asia and the North American Pacific coast, and the populations there are thought to have been introduced from Japan by ship. To test this hypothesis, the genetic divergence among geographic populations of A. togoi was studied using one mitochondrial and three nuclear gene sequences. We detected 71 mitochondrial haplotypes forming four lineages, with high nucleotide diversity around temperate Japan and declining towards peripheral ranges. The major lineage (L1) comprised 57 haplotypes from temperate and subarctic zones in Japan and Southeast Asia including southern China and Taiwan. Two other lineages were found from subtropical islands (L3) and a subarctic area (L4) of Japan. The Canadian population showed one unique haplotype (L2) diverged from the other lineages. In the combined nuclear gene tree, individuals with mitochondrial L4 haplotypes diverged from those with the other mitochondrial haplotypes L1—L3; although individuals with L1—L3 haplotypes showed shallow divergences in the nuclear gene sequences, individuals from Southeast Asia and Canada each formed a monophyletic group. Overall, the genetic composition of the Southeast Asian populations was closely related to that of temperate Japanese populations, suggesting recent gene flow between these regions. The Canadian population might have originated from anthropogenic introduction from somewhere in Asia, but the possibility that it could have spread across the Beringian land bridge cannot be ruled out.     

Osteoprotegerin reduces the serum level of receptor activator of NF-kappaB ligand derived from osteoblasts

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF-kappaB ligand (RANKL). We previously reported that OPG deficiency elevated the circulating level of RANKL in mice. Using OPG(-/-) mice, we investigated whether OPG is involved in the shedding of RANKL by cells expressing RANKL. Osteoblasts and activated T cells in culture released a large amount of RANKL in the absence of OPG. OPG or a soluble form of receptor activator of NF-kappaB (the receptor of RANKL) suppressed the release of RANKL from those cells. OPG- and T cell-double-deficient mice showed an elevated serum RANKL level equivalent to that of OPG(-/-) mice, indicating that circulating RANKL is mainly derived from bone. The serum level of RANKL in OPG(-/-) mice was increased by ovariectomy or administration of 1alpha,25-dihydroxyvitamin D(3). Expression of RANKL mRNA in bone, but not thymus or spleen, was increased in wild-type and OPG(-/-) mice by 1alpha,25-dihydroxyvitamin D(3). These results suggest that OPG suppresses the shedding of RANKL from osteoblasts and that the serum RANKL in OPG(-/-) mice exactly reflects the state of bone resorption.     

Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.     

Visual one-shot learning as an ‘anti-camouflage device’: a novel morphing paradigm

Once people perceive what is in the hidden figure such as Dallenbach’s cow and Dalmatian, they seldom seem to come back to the previous state when they were ignorant of the answer. This special type of learning process can be accomplished in a short time, with the effect of learning lasting for a long time (visual one-shot learning). Although it is an intriguing cognitive phenomenon, the lack of the control of difficulty of stimuli presented has been a problem in research. Here we propose a novel paradigm to create new hidden figures systematically by using a morphing technique. Through gradual changes from a blurred and binarized two-tone image to a blurred grayscale image of the original photograph including objects in a natural scene, spontaneous one-shot learning can occur at a certain stage of morphing when a sufficient amount of information is restored to the degraded image. A negative correlation between confidence levels and reaction times is observed, giving support to the fluency theory of one-shot learning. The correlation between confidence ratings and correct recognition rates indicates that participants had an accurate introspective ability (metacognition). The learning effect could be tested later by verifying whether or not the target object was recognized quicker in the second exposure. The present method opens a way for a systematic production of “good” hidden figures, which can be used to demystify the nature of visual one-shot learning.