Molecular survey of the phosphoserine phosphatase involved in L-serine synthesis by silkworms (Bombyx mori).

Phosphoserine phosphatase (PSP) catalyses the synthesis of l-serine via the phosphorylated pathway by facilitating the dephosphorylation of phosphoserine. A cDNA encoding PSP from the silkworm Bombyx mori (bmPSP) was isolated using reverse transcription-PCR and then sequenced. The resulting clone encoded 236 amino acids with a molecular weight of 26 150, exhibiting 14-60% sequence identity with other PSPs. The recombinant PSP was overexpressed in Escherichia coli and purified. Kinetic studies showed that bmPSP possessed activity toward l-phosphoserine, and Asp20, Asp22 and Asp204 in bmPSP were found to be critical for modulating bmPSP activity. Real-time PCR analysis provided evidence that the amount of bmpsp transcript was reduced in middle silk glands of a sericin-deficient silkworm strain. These findings revealed that bmPSP may play important roles in synthesizing one-carbon donors of l-serine, which is abundant in silk, as well as other cell metabolites in B. mori.

Anisotropy-Induced Soliton Excitation in Magnetized Strong-Rung Spin Ladders.

We report low temperature electron spin resonance experimental and theoretical studies of an archetype S=1/2 strong-rung spin ladder material (C_{5}H_{12}N)_{2}CuBr_{4}. Unexpected dynamics is detected deep in the Tomonaga-Luttinger spin liquid regime. Close to the point where the system is half-magnetized (and believed to be equivalent to a gapless easy plane chain in zero field) we observed orientation-dependent spin gap and anomalous g-factor values. Field theoretical analysis demonstrates that the observed low-energy excitation modes in magnetized (C_{5}H_{12}N)_{2}CuBr_{4} are solitonic excitations caused by Dzyaloshinskii-Moriya interaction presence.

Pebbles and sand on asteroid (162173) Ryugu: In situ observation and particles returned to Earth.

The Hayabusa2 spacecraft investigated the C-type (carbonaceous) asteroid (162173) Ryugu. The mission performed two landing operations to collect samples of surface and subsurface material, the latter exposed by an artificial impact. We present images of the second touchdown site, finding that ejecta from the impact crater was present at the sample location. Surface pebbles at both landing sites show morphological variations ranging from rugged to smooth, similar to Ryugu's boulders, and shapes from quasi-spherical to flattened. The samples were returned to Earth on 6 December 2020. We describe the morphology of >5 grams of returned pebbles and sand. Their diverse color, shape, and structure are consistent with the observed materials of Ryugu; we conclude that they are a representative sample of the asteroid.

Temporal and sequential changes of glial cells and cytokine expression during neuronal degeneration after transient global ischemia in rats.

BACKGROUND: How glial cells and cytokines are associated with the progression of delayed neuronal death induced by transient global ischemia is still unclear. To further clarify this point, we studied morphological changes in glial cells (microglial cells and astrocytes), and cytokine protein levels, during the progression of neuronal cell loss in CA1 (Cornu Ammonis 1) of the hippocampus after transient global ischemia. METHODS: Morphological changes in glial cells were studied immuno-histochemically. Nine cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and TNF-α) were simultaneously measured by a multiplexed bead-based immunoassay from 6 h to day21 after transient four vessel occlusion (4VO) in rats. RESULTS: During the process of neuronal loss, we observed four distinct phases: (1) lag phase day0-2 (no NeuN+ cell loss observed), (2) exponential phase day2-7 (NeuN+ cells reduced in number exponentially), (3) deceleration phase day7-14 (reduction rate of NeuN+ cells became low), (4) stationary phase day14 onward (NeuN+ cell loss progressed no longer). In the lag phase, activated glial cells were observed in the entire hippocampus but later were gradually restricted to CA1. Cytokine protein levels in the lag and exponential phases were lower than in the deceleration and stationary phases. IL-1α, IL-1ß, IL-4, IL-6 and IFN-γ in 4VO were significantly higher in all four phases than in sham. Compared with sham level, GM-CSF was significantly high in the deceleration phase. TNF-α was significantly high in both the deceleration and stationary phases. CONCLUSION: Ischemic stress in 4VO activated glial cells in areas beyond CA1 in the lag phase. Pyramidal neurons were injured in CA1 from the end of the lag phase and then neuronal cells reduced in CA1 in the exponential phase. After neuronal death began, the influence of dead cells on glial cells and cytokine expression gradually became stronger than the influence by ischemic stress. Therefore, from the deceleration phase, changes in glial cells and cytokine production were likely caused by dead cells. Cytokine interaction in the microenvironment may determine the functions of IL-1α, IL-1ß, IL-4, IL-6 and IFN-γ in all four phases. The function of GM-CSF and TNF-α in the deceleration phase may be neurotrophic.

Overcoming the ceiling effects of experts' motor expertise through active haptic training.

One of the most challenging issues among experts is how to improve motor skills that have already been highly trained. Recent studies have proposed importance of both genetic predisposition and accumulated amount of practice for standing at the top of fields of sports and performing arts. In contrast to the two factors, what is unexplored is how one practices impacts on experts' expertise. Here, we show that training of active somatosensory function (active haptic training) enhances precise force control in the keystrokes and somatosensory functions specifically of expert pianists, but not of untrained individuals. By contrast, training that merely repeats the task with provision of error feedback, which is a typical training method, failed to improve the force control in the experts, but not in the untrained. These findings provide evidence that the limit of highly trained motor skills could be overcome by optimizing training methods.

The effects of MPTP on the activation of microglia/astrocytes and cytokine/chemokine levels in different mice strains.

The effects of MPTP on two mouse strains with different MPTP sensitivities and immunological backgrounds were compared: MPTP-sensitive C57BL/6 mice (B6) with a propensity for Th1 and less MPTP-sensitive BALB/c mice (BALB) with a propensity for Th2. It was found that acute MPTP treatment induced behavioral dysfunction, activated microglia/astrocytes, and increased the levels of IL-10, IL-12(p40) IL-13, IFN-gamma, and MCP-1 in CSF in B6, but not in BALB. This suggests that variances in immunological backgrounds might be a major contributing factor in sensitivity differences to MPTP.

Expertise-dependent modulation of muscular and non-muscular torques in multi-joint arm movements during piano keystroke.

The problem of skill-level-dependent modulation in the joint dynamics of multi-joint arm movements is addressed in this study using piano keystroke performed by expert and novice piano players. Using the measured kinematic and key-force data, the time varying net, gravitational, motion-dependent interaction (INT), key-reaction (REA), and muscular (MUS) torques at the shoulder, elbow, wrist, and metacarpophalangeal (MP) joints were computed using inverse dynamics techniques. INTs generated at the elbow and wrist joints, but not those at the MP joint, were greater for the experts as compared with the novices. REA at the MP joint, but not at the other joints, was less for the experts as compared with the novices. The MUSs at the MP, wrist, and elbow joints were smaller, and that at the shoulder joint was larger for the experts as compared with the novices. The experts also had a lesser inter-strike variability of key striking force and key descending velocity as compared with the novices. These findings indicated that the relationship among the INT, REA, and MUS occurring at the joints of the upper-extremity differed between the expert and novice piano players, suggesting that the organization of multi-joint arm movement is modulated by long-term motor training toward facilitating both physiological efficiency and movement accuracy.

Generalized strength of weighted scale-free networks.

An analysis to describe statistical properties of weighted complex networks is proposed. Effective structures of weighted networks depend on how strongly weights w are paid attention or which weights are relevant to the network problem. Defining the metaweight w;{q} with a real parameter q , we characterize systematically weighted complex networks depending on the level of importance of weights. It is found that power-law distribution functions R_{q}[s(q)] of metastrengths s(q) defined by s_{i}(q)= summation operator_{j}w_{ij};{q} , where i and j denote node indices for any q are characterized by only three exponents if the weight distribution is independent of network topology. We also examine the validity of our analytical arguments and the meaning of power-law forms of R_{q}[s(q)] for different q values by illustrating some examples.

Theoretical analysis of effect of tip atomic species on apparent barrier height on Al(100) surface.

We have analyzed the effect of the tip atomic species on the tip-sample separation and the bias-voltage dependence of apparent barrier height (ABH) on an Al(100) surface using the boundary-matching scattering-state density functional method, which can be used to calculate electron states under applied bias voltages self-consistently within the density functional theory. We found that, from the dependence of the tip-sample separation, the difference between measurements with the two tip atomic species is larger in the ABH than in the maximum barrier height evaluated from the calculated potential profile. Furthermore, we found that the bias-polarity dependence of the ABH measured with the Na tip shows behavior opposite to that shown by the ABH measured with the Al tip. These results can be understood from the difference in the degree of lateral confinement of tunneling electrons in the tunneling barrier region between the two atomic species.

The platelet-activating receptor C-type lectin receptor-2 plays an essential role in liver regeneration after partial hepatectomy in mice.

Essentials Regeneration role of C-type lectin receptor-2 (CLEC-2) after 70% hepatectomy (HPx) was investigated. Wild-type or CLEC-2 deleted from platelets of chimeric mice (flKO) underwent HPx. The liver/body weight ratio was significantly lower in the flKO than in the wild-type. CLEC-2 plays an essential role in liver regeneration after HPx. SUMMARY: Background and aim The aim of the present study was to investigate the role of C-type lectin receptor (CLEC)-2 in liver regeneration following partial liver resection in mice. Materials and methods Irradiated chimeric mice transplanted with fetal liver cells from wild-type (WT) mice, CLEC-2-deleted (KO) mice or mice with CLEC-2 deleted specifically from platelets (flKO) were generated. Mice underwent 70% partial hepatectomy (PH). Immunohistochemical staining was performed to investigate the expression of the endogenous ligand for CLEC-2, podoplanin. The accumulation of platelets in the liver was also quantified. The hepatic expression of the IL-6/gp130 and STAT3, Akt and ERK1/2 was also examined. Results The liver/body weight ratio and expression of all cell proliferation markers were significantly lower in the flKO group than in the WT group. The expression of phosphorylated (p) Akt and pERK1/2 was similar in the WT and flKO groups. On the other hand, the expression of pSTAT3 and IL-6 was significantly stronger in the WT group than in the flKO group. The expression of podoplanin was detected in the hepatic sinusoids of both groups. However, the extent to which platelets accumulated in hepatic sinusoids was significantly less in the flKO group than in the WT group. Conclusion CLEC-2 was involved in hepatic regeneration after liver resection and CLEC-2-related liver regeneration was attributed to the interaction between platelets and sinusoidal endothelial cells.

STAT3 integrates cooperative Ras and TGF-ß signals that induce Snail expression.

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during the progression of epithelial tumors. EMT can be induced by transforming growth factor ß (TGF-ß) in certain kinds of cancer cells through the induction of Snail, a key regulator of EMT. We have previously found that TGF-ß remarkably induces Snail expression in cooperation with Ras signals; however, the underlying mechanism of this synergism has not yet been determined. Here, we demonstrate that signal transducer and activator of transcription 3 (STAT3) acts as a mediator that synergizes TGF-ß and Ras signals. The overexpression of STAT3 enhanced Snail induction, whereas siRNA-mediated knockdown of STAT3 inhibited it. The STAT3-YF mutant, which has Tyr 705 substituted with Phe, did not enhance Snail induction. Several STAT3 mutants lacking transcriptional activity also failed to enhance it; however, the putative STAT3-binding elements in the Snail promoter regions were not required for STAT3-mediated Snail induction. Protein inhibitor of activated STAT3 (PIAS3) inhibited the enhanced Snail promoter activity induced by TGF-ß and Ras. The interaction between PIAS3 and STAT3 was reduced by TGF-ß in cells harboring oncogenic Ras, whereas TGF-ß promoted the binding of PIAS3 to Smad3, a crucial mediator of TGF-ß signaling. Therefore, these findings suggest that STAT3 enhances Snail induction when it is dissociated from PIAS3 by TGF-ß in cooperation with Ras signals.

3-Phosphoglycerate dehydrogenase, a key enzyme for l-serine biosynthesis, is preferentially expressed in the radial glia/astrocyte lineage and olfactory ensheathing glia in the mouse brain.

l-Serine is synthesized from glycolytic intermediate 3-phosphoglycerate and is an indispensable precursor for the synthesis of proteins, membrane lipids, nucleotides, and neuroactive amino acids d-serine and glycine. We have recently shown that l-serine and its interconvertible glycine act as Bergmann glia-derived trophic factors for cerebellar Purkinje cells. To investigate whether such a metabolic neuron-glial relationship is fundamental to the developing and adult brain, we examined by in situ hybridization and immunohistochemistry the cellular expression of 3-phosphoglycerate dehydrogenase (3PGDH), the initial step enzyme for de novo l-serine biosynthesis in animal cells. At early stages when the neural wall consists exclusively of the ventricular zone, neuroepithelial stem cells expressed 3PGDH strongly and homogeneously. Thereafter, 3PGDH expression was downregulated and eventually disappeared in neuronal populations, whereas its high expression was transmitted to the radial glia and later to astrocytes in the gray and white matters. In addition, 3PGDH was highly expressed throughout development in the olfactory ensheathing glia, a specialized supporting cell that thoroughly ensheathes olfactory nerves. These results establish a fundamental link of the radial glia/astrocyte lineage and olfactory ensheathing glia to l-serine biosynthesis in the brain. We discuss this finding in the context of the hypothesis that 3PGDH expression in these glia cells contributes to energy metabolism in differentiating and differentiated neurons and other glia cells, which are known to be vulnerable to energy loss.

Astroglial expression of ceramide in Alzheimer's disease brains: a role during neuronal apoptosis.

Accumulating evidences indicate that ceramide is closely involved in apoptotic cell death in neurodegenerative disorders and aging. We examined ceramide levels in the cerebrospinal fluid (CSF) or brain tissues from patients with neurodegenerative disorders and the mechanism of how intra- and extracellular ceramide was regulated during neuronal apoptosis. We screened the ceramide levels in the CSF of patients with neurodegenerative disorders, and found that ceramide was significantly increased in patients with Alzheimer's disease (AD) than in patients with age-matched amyotrophic lateral sclerosis (ALS) and other neurological controls. With immunohistochemistry in AD brains, ceramide was aberrantly expressed in astroglia in the frontal cortices, but not detected in ALS and control brains. To explore for the regulation of ceramide in astroglia in Alzheimer's disease brains, we examined the metabolism of ceramide during neuronal apoptosis. In retinoic acid (RA)-induced neuronal apoptosis, RA slightly increased de novo synthesis of ceramide, but interestingly, RA dramatically inhibited conversion of [14C] ceramide to glucosylceramide (GlcCer), suggesting that the increase of ceramide mass is mainly due to inhibition of the ceramide-metabolizing enzyme GlcCer synthase. In addition, a significant increase of the [14C] ceramide level in the culture medium was detected by chasing and turnover experiments without alteration of extracellular [14C] sphingomyelin levels. A 2.5-fold increase of ceramide mass in the supernatant was also detected after 48 h of treatment with RA. These results suggest a regulatory mechanism of intracellular ceramide through inhibition of GlcCer synthase and a possible role of ceramide as an extracellular/intercellular mediator for neuronal apoptosis. The increased ceramide level in the CSF from AD patients, which may be derived from astroglia, raises a possibility of neuronal apoptosis by the response to intercellular ceramide in AD.

Distinct digit kinematics by professional and amateur pianists.

Many everyday tasks such as typing, grasping, and object manipulation require coordination of dynamic movement across multiple joints and digits. Playing a musical instrument is also one such task where the precise movement of multiple digits is transformed into specific sounds defined by the instrument. Through extensive practice musicians are able to produce precisely controlled movements to interact with the instrument and produce specific sequences of sounds. The present study aimed to determine what aspects of these dynamic movement patterns differ between pianists who have achieved professional status compared to amateur pianists that have also trained extensively. Common patterns of movement for each digit strike were observed for both professional and amateur pianists that were sequence specific, i.e. influenced by the digit performing the preceding strike. However, group differences were found in multi-digit movement patterns for sequences involving the ring or little finger. In some sequences, amateur subjects tended to work against the innate connectivity between digits while professionals allowed slight movement at non-striking digits (covariation) which was a more economical strategy. In other sequences professionals used more individuated finger movements for performance. Thus the present study provided evidence in favor of enhancement of both movement covariation and individuation across fingers in more skilled musicians, depending on fingering and movement sequence.

Neurovascular compression of the rostral ventrolateral medulla related to essential hypertension.

The rostral ventrolateral medulla (RVLM) is thought to serve as a final common pathway for the integration of central cardiovascular information and to be important for the mediation of central pressor responses. An association between essential hypertension and neurovascular compression of the RVLM has been reported. To confirm this relationship and to quantitatively measure the distances between the RVLM and the neighboring arteries, we performed magnetic resonance imaging using a high-resolution 512x512 matrix and magnetic resonance angiography in 49 subjects (21 patients with essential hypertension, 10 patients with secondary hypertension, and 18 normotensive subjects). One patient with essential hypertension was excluded from the evaluations because of inadequate assessment due to poor images. Neurovascular compression of the RVLM was observed in 15 of 20 (75%) patients with essential hypertension. In contrast, neurovascular compression was observed in only 1 of 10 (10%) patients with secondary hypertension and only 2 of 18 (11%) normotensive subjects. The rate of observed neurovascular compression in the essential hypertension group was significantly higher than that in the secondary hypertension group and the normotensive group (P<.01 for both). The distances between the RVLM and the nearest arteries in the essential hypertension group were significantly shorter than those in the other groups (P<.05 for all). On the other hand, the distances between the surface of the medulla oblongata and the nearest arteries did not differ among these three groups. These results suggest that neurovascular compression of the RVLM, but not of the other regions of the medulla oblongata, is particularly related to essential hypertension.

Sympathetic activation and contribution of genetic factors in hypertension with neurovascular compression of the rostral ventrolateral medulla.

OBJECTIVE: The rostral ventrolateral medulla is an important center for the regulation of sympathetic and cardiovascular activities. Reportedly, neurovascular compression of the rostral ventrolateral medulla may be causally related to essential hypertension. We aimed to determine the mechanism behind elevated blood pressure in hypertensive patients with compression of the rostral ventrolateral medulla and to investigate whether genetic factors contribute to the etiology of hypertension with compression. DESIGN AND METHODS: The study included 56 patients with essential hypertension and 25 normotensive individuals. With the use of magnetic resonance imaging, the essential hypertension group was subdivided into hypertension with compression and without compression groups. We compared plasma levels of hormones that raise blood pressure and family histories of hypertension between the two hypertension groups and the normotension group. RESULTS: Plasma norepinephrine levels, but not plasma renin activity, aldosterone, epinephrine, or vasopressin levels, were significantly higher in the hypertension with compression group (389+/-53 pg/ml) than in the hypertension without compression group (217+/-38, P<0.05) or in the normotension group (225+/-30, P<0.05). The percentage of individuals who had two hypertensive parents was significantly higher in the hypertension with compression group (39.4%) than in the hypertension without compression group (13.0%, P<0.05) or in the normotension group (8.0%, P<0.01). CONCLUSIONS: These results indicate that neurovascular compression of the rostral ventrolateral medulla might be, at least in part, causally related to essential hypertension by increasing sympathetic nerve activity. They also suggest that genetic factors might contribute to the etiology of hypertension with neurovascular compression.

Ganglioside/calmodulin kinase II signal inducing cdc42-mediated neuronal actin reorganization.

Cell surface glycoconjugates are thought to mediate cell-cell recognition and play roles in neuronal development and functions. We demonstrated here that exposure of neuronal cells to nanomolar levels of gangliosides Neu5Acalpha 8Neu5Acalpha 3Galbeta 4GlcCer, Galbeta 3GalNAcbeta 4(Neu5Acalpha 8Neu5Acalpha 3)Galbeta 4GlcCer (GD1b), Neu5Acalpha 3Galbeta 3GalNAcbeta 4(Neu5Acalpha 8Neu5Acalpha 3)Galbeta 4GlcCer (GT1b) or its oligosaccharide portion induced a rapid and transient activation of Ca2+/calmodulin-dependent protein kinase II (CaM-KII) in the subplasmalemma. Galbeta 3GalNAcbeta 4(Neu5Acalpha 3)Galbeta 4GlcCer (GM1), GalNAcbeta 4(Neu5Acalpha 3)Galbeta 4GlcCer, Neu5Acalpha 3Galbeta 4GlcCer, Neu5Acalpha 3Galbeta 3GalNAcbeta 4(Neu5Acalpha 3)Galbeta 4GlcCer (GD1a), and Neu5Acalpha 8Neu5Acalpha 3Galbeta 3GalNAcbeta 4(Neu5Acalpha 8Neu5Acalpha 3)-Galbeta 4GlcCer were ineffective. GT1b and GD1b stimulated transient elevation of bulk cytosolic Ca2+ levels while GM1 slightly elevated the levels and GD1a did not. Thus, the cytosolic Ca2+ elevation by the gangliosides may trigger the CaM-KII activation. The treatment was accompanied by peripheral actin polymerization and filopodia formation in NG108-15 cells and primary hippocampal neurons, but not in glial cells. CaM-KII inhibitors blocked both CaM-KII activation and the subsequent filopodia formation. A small G-protein cdc42 was a potential downstream target of CaM-KII activated by the gangliosides. These results suggest that oligosaccharides of the gangliosides serve as potential regulators of the filopodia formation in neuronal cells by triggering the activation of CaM-KII followed by cdc42 up-regulation via a cell surface receptor-like component. The filopodia formation induced by the gangliosides may have a physiological relevance because long-term exposure of hippocampal neurons to GT1b oligosaccharide induced advanced dendritogenesis. Furthermore, exposure of cerebellar neurons to GT1b oligosaccharide facilitated CaM-KII-dependent dendritic outgrowth and branch formation of cerebellar Purkinje neurons, in which actin isoforms were localized to motile structures in dendrites. Thus, the ganglioside/CaM-KII signal plays a role in modulating dendritic morphogenesis by inducing cdc42-mediated actin reorganization.

Extracellular carbohydrate-signal triggering cAMP-dependent protein kinase-dependent neuronal actin-reorganization.

Cell surface glycoconjugates are thought to mediate cell-cell recognition and to play roles in neuronal development and functions. We demonstrated here that exposure of neuronal cells to nanomolar levels of glyco-chains with an N-acetylgalactosamine (GalNAc) residue at the non-reducing termini (GalNAc-S) such as GalNAcbeta4(Neu5Acalpha3)Galbeta4GlcCer (GM2) ganglioside, its oligosaccharide portion, GalNAcbeta4Galbeta4GlcCer (Gg(3)) Cer, GalNAcalpha3GalNAcbeta3Galalpha4Galbeta4GlcCer (Gb(5)) Cer (Forssman hapten) and alpha1-4 linked oligomers of GalNAc, induced a rapid and transient activation of cAMP-dependent protein kinase (PKA) in subplasmalemma. The treatment was accompanied by peripheral actin polymerization and filopodia formation in NG108-15 cells and primary cultured hippocampal neurons, but not in glial cells. A cAMP-dependent protein kinase (PKA) selective inhibitor and an adenylate cyclase inhibitor blocked both PKA activation and the subsequent filopodia formation. A small GTPase cdc42 was a potential downstream target of GalNAc-S-activated PKA. These results suggest that extracellular GalNAc-S serve as potential regulators of the filopodia formation in neuronal cells by triggering the activation of PKA followed by cdc42 up-regulation via a cell surface receptor-like component. Filopodia formation induced by GalNAc-S may have a physiological relevance because long-term exposure to GalNAc-S enhanced F-actin-rich dendrite generation of primary cultured hippocampal neurons, and PKA-dependent dendritic outgrowth and branch formation of primary cultured cerebellar Purkinje neurons, in which actin isoforms were localized to motile structures in dendrites. These findings provide evidence for a novel GalNAc/PKA-signaling cascade in regulating some neuronal maturation.

Expression of substance P receptor in the substantia nigra.

Since the substantia nigra receives abundant substance P innervations but lacks clear evidences about a presence of substance P receptors, expressions for mRNA and protein of substance P receptors were investigated in the rat to resolve this mismatch. Expression levels of substance P receptors mRNA in the substantia nigra pars compacta and reticulata were 37.7 and 24.1% of those in the striatum, respectively, by reverse transcription-polymerase chain reaction (RT-PCR). Substance P receptors mRNA was found in dopamine neurons of the substantia nigra pars compacta by single cell RT-PCR. Ca. 90% of dopamine neurons in the substantia nigra pars compacta were immunoreactive to anti-substance P receptor antibody in the colchicine treated rats. These are the first direct evidence for the existence of substance P receptors in dopamine neurons of the substantia nigra pars compacta.

Characterization of adrenodoxin precursor expressed in Escherichia coli.

The precursor of bovine adrenodoxin (pAd), a mitochondrial protein, was expressed in Escherichia coli. The cloned cDNA of pAd was ligated to an expression vector pET-3d, and silent mutations were introduced into the N-terminal portion of the cDNA in order to increase the expression. The precursor was highly expressed (approximately 20% of the total cell protein) as the inclusion body, and contained an iron-sulfur center as judged from its optical absorption spectra. The inclusion body was solubilized with 7 M urea and pAd was purified in the presence of urea. The purified pAd was efficiently imported into isolated bovine adrenal cortex mitochondria and processed to the mature form. The import reaction required ATP inside the mitochondria in addition to the inner membrane potential, and was strongly inhibited by trypsin treatment of the mitochondria, as in the case of the in vitro translated precursor. It was, however, not dependent on the unfolding activity of the cytosolic factor with extramitochondrial ATP.