sLORETA-qm for interictal MEG epileptic spike analysis: comparison of location and quantity with equivalent dipole estimation.

OBJECTIVE: To determine whether quantitative modification of a standardised low-resolution brain electromagnetic tomography (sLORETA-qm) could be used as a reliable tool for quantitative analysis of magnetoencephalography (MEG) for analysis of the interictal epileptic spike. To verify the performance of sLORETA-qm, magnetic source location and quantity were compared with the equivalent current dipole (ECD) method. METHODS: A total of 50 sources from 10 patients with epilepsy were obtained. Analyses were performed after the MEG data were 3-70 Hz band-pass filtered. Time points for analysis were selected referring to waveform patterns and the isofield contour map. With the same spherical model, source estimation was conducted with two methods of analysis: ECD and sLORETA-qm. Distance from the centre of the spherical model and intensities were compared between the methods. RESULTS: There were no significant differences between the methods in the distance from the spherical model (paired t-test, p=0.8761). Source intensities between the methods were strongly correlated (Spearman's Rho=0.9803, p<0.001). CONCLUSIONS: sLORETA-qm was closely correlated with ECD concerning point source location and quantity in analysis of the interictal epileptic spike. SIGNIFICANCE: sLORETA-qm is a reliable quantifiable method without arbitrariness for analysis of the interictal epileptic spike.

Turn motif phosphorylation negatively regulates activation loop phosphorylation in Akt.

Akt, also known as protein kinase B, has a central role in various signaling pathways that regulate cellular processes such as metabolism, proliferation and survival. On stimulation, phosphorylation of the activation loop (A-loop) and hydrophobic motif (HM) of Akt by the kinase phosphoinositide-dependent kinase 1 (PDK1) and the mammalian target of rapamycin complex 2 (mTORC2), respectively, results in Akt activation. A well-conserved threonine in the turn motif (TM) is also constitutively phosphorylated by mTORC2 and contributes to the stability of Akt. The role of TM phosphorylation in HM and A-loop phosphorylation has not been sufficiently evaluated. Using starfish oocytes as a model system, this study provides the first evidence that TM phosphorylation has a negative role in A-loop phosphorylation. In this system, the maturation-inducing hormone, 1-methyladenine, stimulates Akt to reinitiate meiosis through activation of cyclin B-Cdc2. The phosphorylation status of Akt was monitored via introduction of exogenous human Akt (hAkt) in starfish oocytes. TM and HM phosphorylation was inhibited by microinjection of an anti-starfish TOR antibody, but not by rapamycin treatment, suggesting that both phosphorylation events depend on TORC2, as reported in mammalian cells. A single or double alanine substitution at each of three phosphorylation residues revealed that TM phosphorylation renders Akt susceptible to dephosphorylation on the A-loop. When A-loop phosphatase was inhibited by okadaic acid (OA), TM phosphorylation still reduced A-loop phosphorylation, suggesting that the effect is caused at least partially through reduction of sensitivity to PDK1. Negative regulation by TM phosphorylation was also observed in constitutively active Akt and was functionally reflected in meiosis resumption. By contrast, HM phosphorylation enhanced A-loop phosphorylation and achieved full activation of Akt via a mechanism at least partially independent of TM phosphorylation. These observations provide new insight into the mechanism controlling Akt phosphorylation in the cell.

Improvement of temporal and dynamic subtraction images on abdominal CT using 3D global image matching and nonlinear image warping techniques.

Accurate registration of the corresponding non-enhanced and arterial-phase CT images is necessary to create temporal and dynamic subtraction images for the enhancement of subtle abnormalities. However, respiratory movement causes misregistration at the periphery of the liver. To reduce these misregistration errors, we developed a temporal and dynamic subtraction technique to enhance small HCC by 3D global matching and nonlinear image warping techniques. The study population consisted of 21 patients with HCC. Using the 3D global matching and nonlinear image warping technique, we registered current and previous arterial-phase CT images or current non-enhanced and arterial-phase CT images obtained in the same position. The temporal subtraction image was obtained by subtracting the previous arterial-phase CT image from the warped current arterial-phase CT image. The dynamic subtraction image was obtained by the subtraction of the current non-enhanced CT image from the warped current arterial-phase CT image. The percentage of fair or superior temporal subtraction images increased from 52.4% to 95.2% using the new technique, while on the dynamic subtraction images, the percentage increased from 66.6% to 95.2%. The new subtraction technique may facilitate the diagnosis of subtle HCC based on the superior ability of these subtraction images to show nodular and/or ring enhancement.

A computer-aided temporal and dynamic subtraction technique of the liver for detection of small hepatocellular carcinomas on abdominal CT images.

It is often difficult for radiologists to identify small hepatocellular carcinomas (HCCs) due to insufficient contrast enhancement. Therefore, we have developed a new computer-aided temporal and dynamic subtraction technique to enhance small HCCs, after automatically selecting images set at the same anatomical position from the present (non-enhanced and arterial-phase CT images) and previous images. The present study was performed with CT images from 14 subjects. First, we used template-matching based on similarities in liver shape between the present (non-enhanced and arterial-phase CT images) and previous arterial-phase CT images at the same position. Temporal subtraction images were then obtained by subtraction of the previous image from the present image taken at the same position of the liver. Dynamic subtraction images were also obtained by subtraction of non-enhanced CT images from arterial-phase CT images taken at the same position of the liver. Twenty-one of 22 nodules (95.5%) with contrast enhancement were visualized in temporal and dynamic subtraction images. Compared with present arterial-phase CT images, increases of 150% and 140% in nodule-to-liver contrast were observed on dynamic and temporal subtraction images, respectively. These subtraction images may be useful as reference images in the detection of small moderately differentiated HCCs.

Congenital transverse deficiency of the tibia and fibula: a report of two cases.

We report two similar, but unrelated, patients with congenital bilateral partial deficiencies of the tibia and fibula associated with intact feet. In both patients, the tibia and fibula were absent on initial radiographs, while the femur and the tarsal bones were well developed and there was bilateral teratologic dislocation of the hips. Ultrasound and magnetic resonance imaging (MRI) studies suggested the presence of cartilaginous remnants of the tibia and fibula. There were multidirectional instabilities in the knees and ankles. The clinical and radiological features of these cases are distinct from those of congenital longitudinal deficiency of the tibia, in which the fibula is always preserved, and from longitudinal deficiency of the fibula, in which the tibia is present and the foot is usually involved. We suggest that the bilateral partial deficiencies of the tibia and fibula associated with the intact foot and teratologic dislocation of the hips is a single-entity disorder, possibly categorized as an intercalary transverse deficiency of the lower limb.

Mechanical and dimensional adaptation of rabbit carotid artery cultured in vitro.

The effects of the mechanical environment on arterial walls were investigated in rabbit common carotid arteries, cultured for six days under three different intraluminal pressures (0, 80 and 160 mmHg) in a perfusion culture system. The mechanical responses following the culture were examined using a quasi-static pressure-diameter test. Specimen viability was determined by smooth muscle contraction induced with KCl. Eighteen out of 21 cultured segments showed a peak reduction in diameter of more than 10% and were used for the analysis. The arterial segments cultured at 0 mmHg had a significantly smaller diameter than those cultured at other pressures. The segments cultured at higher pressure had lower incremental elastic moduli at 20 and 80 mmHg and higher moduli at 160 mmHg. The walls of the cultured segments were thicker in groups with higher pressure. These results indicate that, even in culture, the mechanical environment is a major determinant for the mechanical property and dimensions of the arterial wall. Arterial walls may respond to their mechanical environment even if other factors, such as hormonal environment and nervous stimuli, are kept unchanged.

Development and evaluation of the interferometric monitor for greenhouse gases: a high-throughput fourier-transform infrared radiometer for nadir earth observation.

The interferometric monitor for greenhouse gases (IMG) was the precursor of the high-resolution Fourier-transform infrared radiometer (FTIR) onboard a satellite for observation of the Earth. The IMG endured the stress of a rocket launch, demonstrating that the high-resolution, high-throughput spectrometer is indeed feasible for use onboard a satellite. The IMG adopted a newly developed lubricant-free magnetic suspension mechanism and a dynamic alignment system for the moving mirror with a maximum traveling distance of 10 cm. We present the instrumentation of the IMG, characteristics of the movable mirror drive system, and the evaluation results of sensor specifications during space operation.

In vivo regulation of cyclin A/Cdc2 and cyclin B/Cdc2 through meiotic and early cleavage cycles in starfish.

In starfish, fertilization occurs naturally at late meiosis I. In the absence of fertilization, however, oocytes complete meiosis I and II, resulting in mature eggs arrested at the pronucleus stage, which are still fertilizable. In this study, we isolated cDNAs of starfish cyclin A and Cdc2, and monitored extensively the cell cycle dynamics of cyclin A and cyclin B levels and their associated Cdc2 kinase activity, Tyr phosphorylation of Cdc2, and Cdc25 phosphorylation states throughout meiotic and early embryonic cleavage cycles in vivo. In meiosis I, cyclin A was undetectable and cyclin B/Cdc2 alone exhibited histone H1 kinase activity, while thereafter both cyclin A/Cdc2 and cyclin B/Cdc2 kinase activity oscillated along with the cell cycle. Cyclin B-, but not cyclin A-, associated Cdc2 was subjected to regulation via Tyr phosphorylation, and phosphorylation states of Cdc25 correlated with cyclin B/Cdc2 kinase activity with some exceptions. Between meiosis I and II and at the pronucleus stage, cyclin A and B levels remained low, Cdc2 Tyr phosphorylation was undetectable, and Cdc25 remained phosphorylated depending on MAP kinase activity, showing a good correlation between these two stages. Upon fertilization of mature eggs, Cdc2 Tyr phosphorylation reappeared and Cdc25 was dephosphorylated. In the first cleavage cycle, under conditions which prevented Cdc25 activity, cyclin A/Cdc2 was activated with a normal time course and then cyclin B/Cdc2 was activated with a significant delay, resulting in the delayed completion of M-phase. Thus, in contrast to meiosis I, both cyclin A and cyclin B appear to be involved in the embryonic cleavage cycles. We propose that regulation of cyclin A/Cdc2 and cyclin B/Cdc2 is characteristic of meiotic and early cleavage cycles.

[State of bullying in a geriatric hospital].

Recently, reports of bullying and beatings among school children has brought these problems to the public attention. Unfortunately similar problems exist among the aged and institutionalized patients in geriatric hospitals, e.g., covert teasing and bullying of the weak. Therefore, the present status of bullying or being bullied in our geriatric hospital was analyzed and their backgrounds studied to develop clinical countermeasures against bullying. Results of this study were as follows; 1. Nineteen percent of patients were victims of bullying, and patients who had performed the bullying were 3.0%. 2. Of those who were bullied 20.0% wanted to change hospitals, while 60.0% felt they could tolerate the situation or suppress their anger, and 13.3% of the patients who had exhausted their patience took revenge on the bully. 3. Patients who had been bullied tended to be more tenacious and had a high level of depression by Self-rating Depression Scale (SDS). The apparent error rate in these results was 22.5%.

In vivo regulation of the entry into M-phase: initial activation and nuclear translocation of cyclin B/Cdc2.

The cyclin B/Cdc2 complex, Cdc2 kinase governs M-phase. Although the intracomplex modification for its activation in vitro has been described extensively, its regulation in vivo is not so well explained so far. In this article, we will focus on the intracellular regulation of the cyclin B/Cdc2 activity, in particular, how it is initially activated in vivo, how its nuclear translocation is executed specifically at the onset of M-phase, and how the activation and the nuclear translocation are coordinated in the cell. These concerted regulations may determine the appropriate timing for the initiation of M-phase.

Initial triggering of M-phase in starfish oocytes: a possible novel component of maturation-promoting factor besides cdc2 kinase.

G2-phase-arrested immature starfish oocytes contain inactive cdc2 kinase and cdc25 phosphatase, and an inactivator for cdc2 kinase. In this system, we have studied how the regulatory balance is apped toward the initial activation of cdc2 kinase. During the hormone-dependent period (Guerrier, P., and M. Doree, 1975. Dev. Biol. 47:341-348), p34cdc2 and cdc25 protein are already converted, though not fully, to active forms, whereas the inactivators for cdc2 kinase and cdc25 phosphatase are able to exhibit their activities if the hormone were removed. We produced "triggered oocytes," in which due to a neutralizing anticdc25 antibody, the activation of cdc2 kinase is prevented out cdc25 protein is phosphorylated slightly after the maturation-inducing hormonal stimulus. In contrast to control immature oocytes, in triggered oocytes the injected cdc2 kinase is not inactivated, and accordingly the level of cdc2 kinase activity required for meiosis reinitiation is much less. These results imply the presence of a cdc2 kinase activity-independent process(es) that suppresses the inactivator for cdc2 kinase and initially phosphorylates cdc25 protein, although this process is reversible during the initial activation of cdc2 kinase. At the most initial triggering of M-phase, the cdc2 kinase activity-independent process might trip the switch leading to the initial activation of cdc2 kinase. Thereafter, in parallel, the cdc2 kinase-dependent feedback loops described by others may cause further increase in cdc2 kinase activity. We propose that a putative suppressor, which downregulates the inactivator for cdc2 kinase independently of nuclear components, might be a previously unrecognized component of maturation-promoting factor.

Degradation of c-Fos by the 26S proteasome is accelerated by c-Jun and multiple protein kinases.

c-Fos is associated with c-Jun to increase the transcription of a number of target genes and is a nuclear proto-oncoprotein with a very short half-life. This instability of c-Fos may be important in regulation of the normal cell cycle. Here we report a mechanism for degradation of c-Fos. Coexpression of c-Fos and c-Jun in HeLa cells caused marked increase in the instability of c-Fos, whereas v-Fos, the retroviral counterpart of c-Fos, was stable irrespective of the coexpression of c-Jun. Interestingly, deletion of the C-terminal PEST region of c-Fos, which is altered in v-Fos by a frameshift mutation, greatly enhanced its stability, with loss of the effect of c-Jun on its stability. c-Fos synthesized in vitro was degraded by the 26S proteasome in a ubiquitin-dependent fashion. Simple association with c-Jun had no effect on the degradation of c-Fos, but the additions of three protein kinases, mitogen-activated protein kinase, casein kinase II, and CDC2 kinase, resulted in marked acceleration of its degradation by the proteasome-ubiquitin system, though only in the presence of c-Jun. In contrast, v-Fos and c-Fos with a truncated PEST motif were not degraded, suggesting that they escaped from down-regulation by breakdown. These findings indicate a new oncogenic pathway induced by acquisition of intracellular stability of a cell cycle modulatory factor.

Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) by proline-directed protein kinases and its dephosphorylation.

We identified two major substrates for the proline-directed protein kinases--cdc2 kinase and tau protein kinase II (TPKII)--in the cytosol fraction from rat brains. The molecular masses of the proteins were 80 and 46 kDa. Because the 80-kDa protein was phosphorylated by protein kinase C and was heat stable, we examined the possibility that the protein might be myristoylated alanine-rich C kinase substrate (MARCKS). On the basis of a comparison between the properties of the 80-kDa protein and purified MARCKS, we concluded that the 80-kDa protein is indeed MARCKS. The amounts of phosphate incorporated into MARCKS by protein kinase C, cdc2 kinase, and TPKII were 1.7, 1.4, and 0.6 mol/mol of the protein, respectively. Two-dimensional tryptic peptide mapping indicated that phosphorylation sites by protein kinase C and proline-directed protein kinases completely differed. Only the seryl residue was phosphorylated by protein kinase C, whereas both seryl and threonyl residues were phosphorylated by cdc2 kinase and TPKII. Phosphorylation of MARCKS by protein kinase C inhibited the binding to calmodulin, whereas phosphorylation by cdc2 kinase and TPKII significantly increased the binding to calmodulin. The holoenzyme of protein phosphatase 2A dephosphorylated MARCKS that had been phosphorylated by protein kinase C, cdc2 kinase, or TPKII, whereas calcineurin was unable to dephosphorylate it. These results suggest that cdc2 kinase and TPKII regulate the functions of MARCKS in different ways from protein kinase C.

Evidence for cdk5 as a major activity phosphorylating tau protein in porcine brain extract.

The major kinase capable of phosphorylating tau in a porcine brain extract was suggested to be a brain cdc2-like kinase, called cdk5. Tau protein components of microtubules assembled in the brain extract using ATP were phosphorylated to a higher level, and showed a slower electrophoretic mobility than those assembled with GTP. Most of this phosphorylation and electrophoretic mobility shift, that occurred in the brain extract incubated with ATP, were inhibited by butyrolactone I, a specific inhibitor of cdc2 kinase and cdk5. Further, butyrolactone I inhibited phosphorylation of tau exogenously added to the brain extract by approximately 70%. cdk5 purified from porcine brain decreased the electrophoretic mobility of dephosphorylated tau by in vitro phosphorylation of tau to the level present in microtubules polymerized with ATP. cdc2 kinase purified from starfish oocytes also phosphorylated tau and shifted its electrophoretic mobility to an extent greater than that obtained with cdk5. Western blot analysis showed that cdc2 kinase phosphorylated epitopes recognized by SMI31, 33, 34, and tau 1 antibodies in tau proteins , while cdk5 phosphorylated the site recognized by SMI33 (corresponding to phosphorylation at Ser235 in the longest human tau isoform) and partially phosphorylated the tau 1 site. Phosphorylation experiments performed on tau in brain extracts, in the presence of okadaic acid, suggested the presence of both okadaic acid-sensitive and -insensitive phosphatases acting on phosphorylated Ser235. Rat tau that was prepared immediately after decapitation showed a similar phosphorylation state to tau in microtubules polymerized with ATP, suggesting that tau is relatively phosphorylated in vivo.

Dephosphorylation of abnormal sites of tau factor by protein phosphatases and its implication for Alzheimer's disease.

The abnormally phosphorylated forms of tau factor are major constituents of neurofibrillary tangles in Alzheimer's disease brain. In order to investigate protein phosphatases which are related to dephosphorylation of abnormal phosphorylation sites, we examined the dephosphorylation of tau factor phosphorylated by three proline-directed type protein kinases. Tau factor phosphorylated by cdc2 kinase and tau protein kinase II was dephosphorylated by the holoenzyme of protein phosphatase 2A and calcineurin, while either the catalytic subunit of protein phosphatase 2A or protein phosphatase 2C could not catalyze the dephosphorylation. From the kinetic analysis, we concluded that tau factors phosphorylated by the protein kinases serve as good substrates for protein phosphatase 2A and calcineurin. On the other hand, tau factor phosphorylated by glycogen synthase kinase 3 alpha was dephosphorylated by the catalytic subunit of protein phosphatases 2A as well as the holoenzyme of protein phosphatase 2A and calcineurin. It has been reported that serines 199, 202 and 396 according to the numbering of the longest human tau isoform are among the major abnormal phosphorylation sites of tau factor. We synthesized two phosphopeptides which contained phosphoserines 199 and 202 or phosphoserine 396 and prepared the polyclonal antibodies specific for the phosphopeptides. Using these antibodies, we confirmed that the holoenzyme of protein phosphatase 2A and calcineurin could dephosphorylate phosphoserines 199, 202 and 396 in tau factor. The catalytic subunit of protein phosphatase 2A could dephosphorylate phosphoserine 396 but not phosphoserines 199 and 202. Neurofibrillary tangles in Alzheimer's disease brain were immunostained with both antibodies but the normal neurons in the normal aged brains were not. The results suggest that protein phosphatase 2A and calcineurin can be involved in the dephosphorylation of abnormal phosphorylation sites in tau factor and that the dephosphorylation of phosphoserine 396 is differently regulated from phosphoserines 199 and 202.

Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics.

We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.

Pharmacokinetic and clinical pilot study of high-dose intermittent ubenimex treatment in patients with myelodysplastic syndrome.

Ubenimex is an orally active aminopeptidase inhibitor with an immunomodulating action. A pharmacokinetic and clinical pilot study of high-dose intermittent ubenimex was performed in patients with myelodysplastic syndrome. Ubenimex (150 mg/body) was administered orally, twice per day (at a 3 hr interval), twice a week, for at least 8 weeks. The concentrations of ubenimex and p-OH ubenimex, another active metabolite, increased gradually up to 7.1 +/- 4.7 micrograms/ml (mean +/- SD) at 5 hr and 0.25 +/- 0.15 mu/ml at 6 hr. The low percentage (4.2%) of the AUC ratio (p-OH ubenimex/ubenimex) suggested minimal metabolism. The plasma half-life of ubenimex was 2.1 +/- 0.7 hr and the total urinary recovery of both was 71.4% in 48 hr. Among 8 patients clinically studied, one achieved good response and another achieved minor response. These findings suggested good bioavailability, and a certain effectiveness of ubenimex in this administration method, which was worth trying, at least in restricted cases, such as cases with refractory disease or with a poor performance status.

Phosphorylation of a proline-directed kinase motif is responsible for structural changes in myogenin.

Myogenin, a member of the MyoD family which governs skeletal muscle differentiation, was identified as a pair of phosphorylated bands on SDS-PAGE during myogenesis. The slow migrating form was found to be hyperphosphorylated myogenin. In vitro phosphorylation by CDC2 kinase caused a prominent reduction in electrophoretic mobility of myogenin. Furthermore, we demonstrated that phosphorylation of the serine residue at position 43 contributes to the modification of myogenin in vivo and in vitro resulting in the reduction in electrophoretic mobility. We propose here that a CDC2-like proline-directed kinase regulates myogenin activity through its phosphorylation.

Phosphorylation of synthetic vimentin peptides by cdc2 kinase.

Synthetic peptide representing the site Ser-41 in vimentin, Leu-Gly-Ser41-Ala42-Leu-Arg44-Arg-Arg-NH2, and its analogs in which Ala-42 was replaced by various amino acids were tested as substrates for cdc2 kinase. Among them, the analog containing sarcosine as well as proline was an excellent substrate. The result suggests that the N-substituted structure of proline immediately following the site is important for cdc2 kinase phosphorylation. Replacement of Ala-42 by polar amino acids, especially lysine, had negative effects on peptide phosphorylation. The peptides in this study were also assayed with another type of proline-directed protein kinase, tau protein kinase II. The substrate specificity differed essentially from that of cdc2 kinase.