Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli

The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III synthase. Although the role of uro'gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro'gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832-5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity.     

Topogenesis and Homeostasis of Fatty Acyl-CoA Reductase 1

Peroxisomal fatty acyl-CoA reductase 1 (Far1) is essential for supplying fatty alcohols required for ether bond formation in ether glycerophospholipid synthesis. The stability of Far1 is regulated by a mechanism that is dependent on cellular plasmalogen levels. However, the membrane topology of Far1 and how Far1 is targeted to membranes remain largely unknown. Here, Far1 is shown to be a peroxisomal tail-anchored protein. The hydrophobic C terminus of Far1 binds to Pex19p, a cytosolic receptor harboring a C-terminal CAAX motif, which is responsible for the targeting of Far1 to peroxisomes. Far1, but not Far2, was preferentially degraded in response to the cellular level of plasmalogens. Experiments in which regions of Far1 or Far2 were replaced with the corresponding region of the other protein showed that the region flanking the transmembrane domain of Far1 is required for plasmalogen-dependent modulation of Far1 stability. Expression of Far1 increased plasmalogen synthesis in wild-type Chinese hamster ovary cells, strongly suggesting that Far1 is a rate-limiting enzyme for plasmalogen synthesis.     

Highly efficient supramolecular photocatalysts for CO2 reduction using visible light.

We report the most efficient homogeneous photocatalyst yet for CO(2) reduction using a wide range of visible-light wavelength. We synthesized new Ru(II)-Re(I) binuclear complexes with 1,3-bis(4'-methyl-[2,2']bipyridinyl-4-yl)-propan-2-ol (bpyC3bpy) as a bridge ligand, specifically [Ru-ReP(OEt)(3)](3+) and [Ru-Repy](3+) where a P(OEt)(3) or pyridine ligand coordinates on the Re site. Their photocatalytic activities were compared with [Ru-ReCl](2+), which has a Cl(-) ligand on the Re site and has recently been reported as a much better photocatalyst (Phi = 0.12, TN(CO) = 160) than a 1:1 mixed system of the corresponding Ru(II) and Re(I) mononuclear complexes. The best photocatalyst was [Ru-ReP(OEt)(3)](3+), for which Phi = 0.21 and TN(CO) = 232. A mechanistic study clearly showed that [Ru-ReP(OEt)(3)](3+) is rapidly converted into the solvento complex [Ru-ReSol](3+), (Sol = DMF or TEOA) which is the actual photocatalyst. Although similar rapid ligand substitution occurs with other supramolecules, the pyridine and Cl(-) anions accelerate the decomposition of the supramolecular photocatalysts.     

Bright-light exposure during daytime sleeping affects nocturnal melatonin secretion after simulated night work

The guidelines for night and shift workers recommend that after night work, they should sleep in a dark environment during the daytime. However, staying in a dark environment during the daytime reduces nocturnal melatonin secretion and delays its onset. Daytime bright-light exposure after night work is important for melatonin synthesis the subsequent night and for maintaining the circadian rhythms. However, it is not clear whether daytime sleeping after night work should be in a dim- or a bright-light environment for maintaining melatonin secretion. The aim of this study, therefore, was to evaluate the effect of bright-light exposure during daytime sleeping on nocturnal melatonin secretion after simulated night work. Twelve healthy male subjects, aged 24.8 ± 4.6 (mean ± SD), participated in 3-day sessions under two experimental conditions, bright light or dim light, in a random order. On the first day, the subjects entered the experimental room at 16:00 and saliva samples were collected every hour between 18:00 and 00:00 under dim-light conditions. Between 00:00 and 08:00, they participated in tasks that simulated night work. At 10:00 the next morning, they slept for 6 hours under either a bright-light condition (>3000 lx) or a dim-light condition (<50 lx). In the evening, saliva samples were collected as on the first day. The saliva samples were analyzed for melatonin concentration. Activity and sleep times were recorded by a wrist device worn throughout the experiment. In the statistical analysis, the time courses of melatonin concentration were compared between the two conditions by three-way repeated measurements ANOVA (light condition, day and time of day). The change in dim light melatonin onset (ΔDLMO) between the first and second days, and daytime and nocturnal sleep parameters after the simulated night work were compared between the light conditions using paired t-tests. The ANOVA results indicated a significant interaction (light condition and3 day) (p = .006). Post hoc tests indicated that in the dim-light condition, the melatonin concentration was significantly lower on the second day than on the first day (p = .046); however, in the bright-light condition, there was no significant difference in the melatonin concentration between the days (p = .560). There was a significant difference in ΔDLMO between the conditions (p = .015): DLMO after sleeping was advanced by 11.1 ± 17.4 min under bright-light conditions but delayed for 7.2 ± 13.6 min after sleeping under dim-light conditions. No significant differences were found in any sleep parameter. Our study demonstrated that daytime sleeping under bright-light conditions after night work could not reduce late evening melatonin secretion until midnight or delay the phase of melatonin secretion without decreasing the quality of the daytime sleeping. Thus, these results suggested that, to enhance melatonin secretion and to maintain their conventional sleep–wake cycle, after night work, shift workers should sleep during the daytime under bright-light conditions rather than dim-light conditions.     

cis‐Regulatory analysis for later phase of anterior neuroectoderm‐specific foxQ2 expression in sea urchin embryos

The specification of anterior neuroectoderm is controlled by a highly conserved molecular mechanism in bilaterians. A forkhead family gene, foxQ2, is known to be one of the pivotal regulators, which is zygotically expressed in this region during embryogenesis of a broad range of bilaterians. However, what controls the expression of this essential factor has remained unclear to date. To reveal the regulatory mechanism of foxQ2, we performed cis‐regulatory analysis of two foxQ2 genes, foxQ2a and foxQ2b, in a sea urchin Hemicentrotus pulcherrimus. In sea urchin embryos, foxQ2 is initially expressed in the entire animal hemisphere and subsequently shows narrower expression restricted to the anterior pole region. In this study, as a first step to understand the foxQ2 regulation, we focused on the later restricted expression and analyzed the upstream cis‐regulatory sequences of foxQ2a and foxQ2b by using the constructs fused to short half‐life green fluorescent protein. Based on deletion and mutation analyses of both foxQ2, we identified each of the five regulatory sequences, which were 4–9 bp long. Neither of the regulatory sequences contains any motifs for ectopic activation or spatial repression, suggesting that later mRNA localization is regulated in situ. We also suggest that the three amino acid loop extension‐class homeobox gene Meis is involved in the maintenance of foxQ2b, the expression of which is dominant during embryogenesis.     

The origin of ascophoran bryozoans was historically contingent but likely

The degree to which evolutionary outcomes are historically contingent remains unresolved, with studies at different levels of the biological hierarchy reaching different conclusions. Here we examine historical contingency in the origin of two evolutionary novelties in bryozoans, a phylum of colonial animals whose fossil record is as complete as that of any major group. In cheilostomes, the dominant living bryozoans, key innovations were the costal shield and ascus, which first appeared in the Cretaceous 85–95 Myr ago. We establish the parallel origin of these structures less than 12 Myr ago in an extant bryozoan genus, Cauloramphus, with transitional stages remarkably similar to those inferred for a Cretaceous clade. By one measure, long lag times in the first origins of costal shield and ascus suggest a high degree of historical contingency. This, however, does not equate with dependence on a narrow set of initial conditions or a low probability of evolution. More than one set of initial conditions may lead to an evolutionary outcome, and alternative sets are not entirely independent. We argue that, although historically contingent, the origin of ascus and costal shield was highly likely with sufficient possibilities afforded by time.     

Tyrosine phosphorylation is involved in Ca(2+)entry in human gingival fibroblasts

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.     

“Sensory Switching” in Elbow Reconstruction

In the treatment of the soft tissue defect of the elbow, flap reconstruction is necessitated in many cases because of thinness of soft tissue at this region. In addition, reacquirement of tactile sensation is desirable because of the anatomical and specific functions of the elbow. Of three cases treated for elbow defects, one was reconstructed with a pedicled island forearm flap containing the lateral cutaneous nerve of the forearm, another was reconstructed with a venoneuro-accompanying artery fasciocutaneous flap (VNAF flap) containing the basilic vein, and the third with the VNAF flap containing the cephalic vein. The three cases demonstrated a sudden change of sensory territory 4 to 6 months after surgery, which was confirmed by touching the reconstructed region with patients'' eye-closed: from its original territory to the elbow in a “switching”-like action. Here we describe and discuss the concept of “sensory switching.”     

Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ‐lyase based on substrate‐assisted catalysis

Methionine γ‐lyse (MGL) catalyzes the α, γ‐elimination of l ‐methionine and its derivatives as well as the α, β‐elimination of l ‐cysteine and its derivatives to produce α‐keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site‐directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51–Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5′‐phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate‐assisted catalysis.