Cloning and expression of the ferredoxin gene from extremely halophilic archaeon Haloarcula japonica strain TR-1

The gene encoding a ferredoxin (Fd) from Haloarcula japonica strain TR-1 was cloned and sequenced. Sequence analysis of the cloned Ha. japonica Fd gene revealed that the structural gene consisted of an open reading frame of 387 nucleotides encoding 129 amino acids. The deduced amino acid sequence of Ha. japonica Fd showed 84 to 98% identity with corresponding sequences in other extremely halophilic archaea. The Ha. japonica Fd gene was inserted into the shuttle vector pWL102 and used to transform Ha. japonica. Ha. japonica Fd could then be produced as a fusion with HisTag (6xHis) in Ha. japonica host cells. The absorption and ESR spectra of the Fd/HisTag fusion protein revealed the presence of a [2Fe-2S] cluster which is characteristic of native Ha. japonica Fd.     

Hemin (Fe(3+))-- and heme (Fe(2+))--smectite conjugates as a model of hemoprotein based on spectrophotometry

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) in acetone to form a hemin-smectite conjugate. The hemin-smectite conjugate became soluble in water to form a transparent colloidal solution with a dark brown color. Its absorption spectrum in water showed a sharp Soret band at 398 nm with the molar extinction coefficient as epsilon(398nm) = 11.6 x 10(4) M(-1) cm(-1), which is in good agreement with epsilon(398nm) = (12.2 +/- 3) x 10(4) M(-1) cm(-1) of monomeric hematin (1). Hemin (Fe(3+))-smectite conjugate had a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and guaiacol (a hydrogen donor) in aqueous solution and its activity was higher than that of hematin. Hemin (Fe(3+))-smectite conjugate in water was reduced by adding sodium dithionite to form a heme (Fe(2+))-smectite conjugate which is also a transparent colloidal solution in water. Its absorption spectrum in aqueous solution was surprisingly in close agreement with that of oxyhemoglobin. Its peak positions of alpha, beta, and Soret bands were located in only a 9--3 nm shift to shorter wavelengths in comparison with those of oxyhemoglobin. Therefore, heme (Fe(2+))-smectite conjugate was bound to O(2) to form O(2)-heme (Fe(2+))-smectite conjugate. The addition of carbon monoxide, CO, to O(2)-heme (Fe(2+))-smectite conjugate caused the formation of CO-heme (Fe(2+))-smectite conjugate with a similar absorption spectrum of carboxyhemoglobin (HbCO) accompanied by shifting 8--10 nm to shorter wavelength. Therefore, the transformation of O(2)-heme (Fe(2+))-smectite conjugate to CO-heme (Fe(2+))-smectite conjugate was accompanied by shifting of 7, 4, and 3 nm to shorter wavelengths in the alpha, beta, and Soret bands respectively, which are similar to the spectral change from oxyhemoglobin to carboxyhemoglobin. Also the ratio (1:1.6) of the molar extinction coefficient of Soret band of O(2)-heme (Fe(2+))-smectite conjugate and CO-heme (Fe(2+))-smectite conjugate was surprisingly agreement with ratio (1:1.5) of oxyhemoglobin and carboxyhemoglobin. The phenomenon shown above was unexpectedly found during the course of study of bioconjugate of a bioactive substance, hemin (Fe(3+)) or heme (Fe(2+)), and a clay mineral, smectite, in place of the protein of globin in hemoglobin.     

PEN-2 enhances gamma-cleavage after presenilin heterodimer formation

The presenilin (PS) complex, including PS, nicastrin, APH-1 and PEN-2, is essential for gamma-secretase activity, which is required for amyloid beta-protein (Abeta) generation. However, the precise individual roles of the three cofactors in the PS complex in Abeta generation remain to be clarified. Here, to distinguish the roles of PS cofactors in gamma-secretase activity from those in PS endoproteolysis, we investigated their roles in the gamma-secretase activity reconstituted by the coexpression of PS N- and C-terminal fragments (NTF and CTF) in PS-null cells. We demonstrate that the coexpression of PS1 NTF and CTF forms the heterodimer and restores Abeta generation in PS-null cells. The generation of Abeta was saturable at a certain expression level of PS1 NTF/CTF, while the overexpression of PEN-2 alone resulted in a further increase in Abeta generation. Although PEN-2 did not enhance PS1 NTF/CTF heterodimer formation, PEN-2 expression reduced the IC50 of a specific gamma-secretase inhibitor, a transition state analogue, for Abeta generation, suggesting that PEN-2 expression enhances the affinity or the accessibility of the substrate to the catalytic site. Thus, our results strongly suggest that PEN-2 is not only an essential component of the gamma-secretase complex but also an enhancer of gamma-cleavage after PS heterodimer formation.     

Reduction of methemoglobin via electron transfer from photoreduced flavin: restoration of O2-binding of concentrated hemoglobin solution coencapsulated in phospholipid vesicles

Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH(2) by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its O(2) binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Phi) of 0.17, that is higher than the method of direct photoexcitation of heme (Phi = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O(2) binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 microm (close to capillary diameter in tissue, 5 microm), and visible light irradiation (221 mW/cm(2)) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH(2) reacted with O(2) to produce H(2)O(2), which was detected by the fluorescence measurement of the reaction of H(2)O(2) and p-nitrophenylacetic acid. However, the amount of H(2)O(2) generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH(2) was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.     

Polycomb group gene mel-18 regulates early T progenitor expansion by maintaining the expression of Hes-1, a target of the Notch pathway

Polycomb group (PcG) proteins play a role in the maintenance of cellular identity throughout many rounds of cell division through the regulation of gene expression. In this report we demonstrate that the loss of the PcG gene mel-18 impairs the expansion of the most immature T progenitor cells at a stage before the rearrangement of the TCR beta-chain gene in vivo and in vitro. This impairment of these T progenitors appears to be associated with increased susceptibility to cell death. We also show that the expression of Hes-1, one of the target genes of the Notch signaling pathway, is drastically down-regulated in early T progenitors isolated from mel-18(-/-) mice. In addition, mel-18(-/-) T precursors could not maintain the Hes-1 expression induced by Delta-like-1 in monolayer culture. Collectively, these data indicate that mel-18 contributes to the maintenance of the active state of the Hes-1 gene as a cellular memory system, thereby supporting the expansion of early T progenitors.     

Effects of cyclohexenonic long-chain fatty alcohol on diabetic rat trachea

In order to investigate the diabetes-associated neuropathy and prevent effects of cyclohexenonic long-chain fatty alcohol, a neurotrophic substance, in trachea, we studied its effect on streptozotocin-diabetic hyper-reactivity in the rat trachea. Diabetes was induced in 8-week-old male Sprague-Dawley rats by administering an intraperitoneal injection of streptozotocin (50 mg/kg). The rats were divided randomly into four groups and were maintained for four weeks: age-matched control rats, diabetic rats without treatment with cyclohexenonic long-chain fatty alcohol, and diabetic rats treated with cyclohexenonic long-chain fatty alcohol (2 and 8 mg/kg, i.p. every day). The serum glucose and insulin levels were determined, and the contractile responses of the trachea induced by carbachol and KCl were investigated. Treatment with cyclohexenonic long-chain fatty alcohol did not alter the rats' diabetic status, i.e., body weight, thickness of the trachea, serum glucose levels, and serum insulin levels, but significantly improved the diabetic-induced hyper-reactivity of the rat trachea in a dose-dependent manner. There was no significant difference in either the carbachol- or KCl-induced contractile forces between groups with or without mucosa in the functional studies. In histological examinations, thinning of cricoid cartilage, thickness of basal membrane, and degeneration, fragmentation of elastic fibers in the submucosal layer, and hypertrophy of smooth muscle bundle in the membranous wall of trachea were observed in the diabetic rat trachea, which were improved by treatment with cyclohexenonic long-chain fatty alcohol. Our data indicate that this drug can prevent hyper-reactivity in the diabetic trachea.     

Identification and characterization of a mouse dipeptidase that hydrolyzes L-carnosine

L-Carnosine is a bioactive dipeptide present in mammalian tissues including the central nervous system. We have recently shown that L-carnosine is involved in the regulation of energy homeostasis through the autonomic nervous system, but the mechanisms for its biosynthesis and degradation have not yet been fully elucidated. Here we report the biochemical and immunohistochemical characterization of a mammalian protein that has a 17% overall amino acid sequence homology with a Lactobacilus carnosinase, PepV. A recombinant protein expressed in E. coli has the enzymatic ability to digest L-carnosine and various other dipeptides, and this activity is inhibited by bestatin. It requires Mn2+ for enzymatic activity and its effect is reversible. Immunohistochemical analysis showed that a few neuronal populations express this protein at very high levels. It is highly expressed in the parafascicular nucleus of the thalamus, tuberomammillary nucleus of the hypothalamus and the mitral cell layer of the olfactory bulb. In addition, neuronal processes, but not cell bodies, are stained in the striatum. In all these areas, the protein did not colocalize with the glial fibrilary acidic protein. These results suggest that a peptidase that digests L-carnosine is enriched in several specific neuronal populations in the central nervous system.     

Regulation of biological activity and matrix assembly of laminin-5 by COOH-terminal, LG4-5 domain of alpha3 chain

The basement membrane protein laminin-5 (LN5; alpha3beta3gamma2) undergoes specific proteolytic processing of the 190-kDa alpha3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4-5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa alpha3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type alpha3 chain was completely cleaved, whereas the mutated alpha3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4-5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa alpha3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4-5 from the 190-kDa alpha3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4-5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4-5 synergistically enhances integrin signaling as it is released from the precursor LN5.     

Crystal structures of the signal transducing protein GlnK from Thermus thermophilus HB8

The Thermus thermophilus HB8 genome encodes a signal transducing PII protein, GlnK. The crystal structures of GlnK have been determined in two different space groups, P2(1)2(1)2(1) and P3(1)21. The PII protein has the T-loop, which is essential for interactions with receptor proteins. In both crystal forms, three GlnK molecules form a trimer in the asymmetric unit. In one P2(1)2(1)2(1) crystal form, the three T-loops in the trimer are disordered, while in another P2(1)2(1)2(1) crystal form, the T-loop from one molecule in the trimer is ordered. In the P3(1)21 crystal, one T-loop is ordered while the other two T-loops are disordered. The conformations of the ordered T-loops significantly differ between the two crystal forms; one makes the alpha-helix in the middle of the T-loop, while the other has an extension of the beta-hairpin. Two different conformations are captured by the crystal contacts. The observation of multiple T-loop conformations suggests that the T-loop could potentially exhibit "polysterism," which would be important for interactions with receptor proteins. The crystal structures of the nucleotide-bound forms, GlnK.ATP and GlnK.ADP, have also been determined. ATP/ADP binding within a cleft at the interface of two adjacent T. thermophilus GlnK monomers might affect the conformation of the T-loop.