Immunohistochemical localization of anabolic and catabolic enzymes for anandamide and other putative endovanilloids in the hippocampus and cerebellar cortex of the mouse brain.

An increasing body of evidence indicates that: 1) the endocannabinoid anandamide (AEA) and other unsaturated N-acylethanolamines (NAEs), 2) 12-(S)-lipoxygenase (12-LOX) products of arachidonic acid, and 3) unsaturated N-acyldopamines (NADAs), act as endogenous ligands of transient receptor potential vanilloid type 1 (TRPV1) channels at intracellular binding sites. AEA is synthesized and released "on demand" in neurons from its membrane precursor, N-arachidonoyl-phosphatidylethanolamine, by an N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), and is inactivated by intracellular hydrolysis by fatty acid amide hydrolase (FAAH), whereas catechol-O-methyl-transferase (COMT) was suggested to inactivate NADAs. However, it is not known whether these enzymes or 12-LOX co-localize to any extent with TRPV1 receptors in the brain. In this study we used immunohistochemical techniques (single peroxidase and double immunofluorescence staining), and analyzed the localization of the TRPV1 channel in mouse hippocampal and cerebellar neurons with respect to NAPE-PLD, FAAH, 12-LOX and COMT. Cycloxygenase-2 (COX-2), another putative AEA-degrading enzyme, was also studied. Co-localization between TRPV1 and either NAPE-PLD or FAAH, COX-2, 12-LOX and COMT was found in Ammon's horn (CA3) hippocampal pyramidal neurons and (with the exception of 12-LOX) in some Purkinje cells. At the cellular level, both anabolic and catabolic enzymes appeared as fine grains with immunoperoxidase labeling and were observed in the somatodendritic compartment of CA3 pyramidal cells as well as (with the exception of 12-LOX) in the cytoplasm of Purkinje neurons, in which FAAH and COX-2 immunoreactivities were, however, preferentially localized in the large extension of the dendritic arbor. Our data agree with the hypothesis that, in potential "endovanillergic" neurons, endogenous TRPV1 agonists, and AEA in particular, act as intracellular mediators by being produced from and/or degraded by the same mouse brain cells that express TRPV1 receptors.

[Investigation of the accurate measurement of the basic imaging properties for the digital radiographic system based on flat panel detector].

PURPOSE/AIM OF THE EXHIBIT: The purpose of this exhibit is: 1. To explain "resampling", an image data processing, performed by the digital radiographic system based on flat panel detector (FPD). 2. To show the influence of "resampling" on the basic imaging properties. 3. To present accurate measurement methods of the basic imaging properties of the FPD system. CONTENT ORGANIZATION: 1. The relationship between the matrix sizes of the output image and the image data acquired on FPD that automatically changes depending on a selected image size (FOV). 2. The explanation of the image data processing of "resampling". 3. The evaluation results of the basic imaging properties of the FPD system using two types of DICOM image to which "resampling" was performed: characteristic curves, presampled MTFs, noise power spectra, detective quantum efficiencies. CONCLUSION/SUMMARY: The major points of the exhibit are as follows: 1. The influence of "resampling" should not be disregarded in the evaluation of the basic imaging properties of the flat panel detector system. 2. It is necessary for the basic imaging properties to be measured by using DICOM image to which no "resampling" is performed.

M phase-specific kinetochore proteins in fission yeast: microtubule-associating Dis1 and Mtc1 display rapid separation and segregation during anaphase.

BACKGROUND: Kinetochore microtubules are made early in mitosis and link chromosomal kinetochores to the spindle poles. They are required later to move the separated sister chromatids toward the opposite poles upon the onset of anaphase. Very little is known about proteins that are responsible for the connection between kinetochores and mitotic microtubules. RESULTS: We here show that fission yeast Dis1 and the related protein Mtc1/Alp14 are both able to bind microtubules in vitro and share an essential function for viability in vivo. The deletion of mtc1+ results in an instability of cytoplasmic microtubules that can be suppressed by the ectopic expression of dis1+. Dis1 and Mtc1 are localized along interphase cytoplasmic microtubules and are mobilized onto the spindle upon mitotic commitment. In chromatin immunoprecipitation (CHIP) experiments Dis1 coprecipitated with the central centromeric DNA in an M phase-specific manner. Consistently, observations of both living cells in which the native, genomic copy of dis1+ tagged with GFP and cells fixed by immunostaining established that Dis1 behaves as a kinetochore protein during the progression from metaphase to anaphase. The central and C-terminal regions of Dis1 are sufficient for interactions with microtubules and the kinetochore, respectively. In anaphase, the GFP signals of both Dis1 and Mtc1 suddenly separate and move quickly toward opposite spindle poles. CONCLUSIONS: Fission yeast Dis1 and Mtc1 are members of an evolutionarily conserved microtubule binding protein family that includes frog XMAP215. Dis1 and Mtc1 are implicated in stabilizing kinetochore microtubules in metaphase and so counteract the action of microtubule destabilizing factors that dominate in anaphase. Dis1 may play a dual role by becoming a part of the kinetochores in an M phase-specific manner, and it may possibly generate connections between kinetochores and microtubules.

Changes in cholinergic function in the frontal cortex and hippocampus of rat exposed to ethanol and acetaldehyde.

Our previous microdialysis study demonstrated that both ethanol (EtOH) and acetaldehyde (ACe) decrease in vivo acetylcholine (ACh) release in the medial frontal cortex of freely moving rats. To better understand the mechanisms of EtOH and ACe's effects on the cholinergic system in the brain, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) expression was examined at 40 and 240 min after a dose of EtOH (1 g/kg) in the rat frontal cortex and hippocampus. The control group was treated with 0.9% saline, and other groups received EtOH or cyanamide (CY, 50 mg/kg, a potent aldehyde dehydrogenase inhibitor) and 60 min later by EtOH intraperitoneally. Reverse-transcription polymerase chain reaction (RT-PCR) analysis revealed that ChAT mRNA levels were decreased by 72.8% and 71.6% in the EtOH and CY+EtOH groups, respectively, at 40 min after EtOH injection compared with saline in the frontal cortex. The hippocampal ChAT levels were reduced by 76.5% and 53.0% in the EtOH and CY+EtOH groups, respectively, at this time. CY+EtOH-induced depletion in ChAT mRNA levels was markedly higher than EtOH in the hippocampus. A similar decrease pattern of ChAT was observed at protein levels as determined by Western blot, but the reduced ChAT levels were significantly higher in the CY+EtOH group as compared with the EtOH group both in the frontal cortex and hippocampus. At 240 min after EtOH injection, the EtOH group had no effect on ChAT at mRNA levels, as compared with saline, whereas CY+EtOH group induced a significant decrease in ChAT mRNA expression to 62.0% and 65.5% in the frontal cortex and hippocampus, respectively. These data were consistent with the results of the Western blot analysis. AChE expression at mRNA levels was not changed at either 40 or 240 min after EtOH dosing in either of these groups in the frontal cortex and hippocampus. Within 40 and 240 min, a statistically significant difference in ChAT expression at mRNA and protein levels was found in the EtOH and CY+EtOH groups both in the frontal cortex and hippocampus. The data obtained from this study demonstrate that EtOH and ACe concentrations decreased ChAT expression at 40 and 240 min after EtOH administration in the frontal cortex and hippocampus, and this result suggests that reduced ChAT expression is strongly related to a decrease in ACh release in the rat brain.

An automated patient recognition method based on an image-matching technique using previous chest radiographs in the picture archiving and communication system environment.

An automated patient recognition method for correcting "wrong" chest radiographs being stored in a picture archiving and communication system (PACS) environment has been developed. The method is based on an image-matching technique that uses previous chest radiographs. For identification of a "wrong" patient, the correlation value was determined for a previous image of a patient and a new, current image of the presumed corresponding patient. The current image was shifted horizontally and vertically and rotated, so that we could determine the best match between the two images. The results indicated that the correlation values between the current and previous images for the same, "correct" patients were generally greater than those for different, "wrong" patients. Although the two histograms for the same patient and for different patients overlapped at correlation values greater than 0.80, most parts of the histograms were separated. The correlation value was compared with a threshold value that was determined based on an analysis of the histograms of correlation values obtained for the same patient and for different patients. If the current image is considered potentially to belong to a "wrong" patient, then a warning sign with the probability for a "wrong" patient is provided to alert radiology personnel. Our results indicate that at least half of the "wrong" images in our database can be identified correctly with the method described in this study. The overall performance in terms of a receiver operating characteristic curve showed a high performance of the system. The results also indicate that some readings of "wrong" images for a given patient in the PACS environment can be prevented by use of the method we developed. Therefore an automated warning system for patient recognition would be useful in correcting "wrong" images being stored in the PACS environment.

Computer-aided diagnosis for interstitial infiltrates in chest radiographs: optical-density dependence of texture measures.

We have been developing a computerized scheme for automated detection and characterization of interstitial infiltrates based on the Fourier transform of lung texture. To improve the performance of the scheme, which was developed using digitized screen-film radiographs, optical-density dependence of both the gradient of the film used and the system noise associated with the laser scanner were investigated. Two hundred chest radiographs, including 100 abnormal cases with interstitial infiltrates, were digitized using a laser scanner. The root-mean-square (RMS) variations and the first moments of the power spectra, which correspond to the magnitude and coarseness of lung texture, were determined by Fourier transform of lung textures in numerous regions of interest (ROIs). The RMS variation was dependent upon the average optical density in the ROI, though no obvious trend existed for the first moment of the power spectrum. Dependence of the RMS variations on optical density was corrected for using the gradient curve of the film. Also, system noise associated with the laser scanner was corrected. Results indicated that the specificity was improved from 81% (without correction) to 89% (with corrections), without any loss of sensitivity (90%). Thus, the correspondence between the computer output and consensus interpretation of radiologists was improved with the new scheme compared to the previous one. This improved computerized scheme may be useful to radiologists in detecting interstitial infiltrates in chest radiographs.

Evaluation of an asymmetric screen-film system for chest radiography.

To evaluate the potential utility of an asymmetric screen-film system for chest radiography, its image quality and detail visibility compared with a conventional screen-film system are investigated. The basic imaging properties were evaluated by measuring Hurter and Driffield curves, resolution properties, and noise Wiener spectra. The visibility of simulated anatomical and pathological details in radiographs of a chest phantom and normal anatomy in chest radiographs of patients were evaluated subjectively. The dynamic range of each system is comparable, though the asymmetric screen-film system can provide an advantage over the conventional system due to a relative dose reduction of approximately 35% and higher resolution properties at high optical densities. The noise level of the asymmetric screen-film system is slightly greater at low optical densities and much greater at high optical densities. However, the visibility of lung details with the asymmetric screen-film system is slightly superior to the conventional screen-film system despite the increase in noise. Mediastinal and retrodiaphragmatic details are similar, though marginally superior with the asymmetric screen-film system. It is concluded that the asymmetric screen-film system provided slightly superior image quality to the conventional screen-film system for chest radiography, provided the average lung density is maintained at a higher level than is customary with conventional systems.

Basic imaging properties of a computed radiographic system with photostimulable phosphors.

We measured the characteristic curve, modulation transfer function (MTF), and the Wiener spectrum of a commercially available computed radiographic (CR) system with photostimulable phosphor plate (imaging plate, IP). The characteristic curve (system response) obtained by an inverse-square x-ray sensitometry showed a wide dynamic range (order of 10(3) in maximum). The slit technique was employed to determine the MTF's, such as IP MTF, presampling MTF including the unsharpness of the detector (IP) and the blurring effect of the sampling aperture, and laser-printer MTF. It was found that the MTF of the standard type of IP was comparable to that of medium-speed screen/film systems. The noticeable degradation of resolution in our CR system, however, occurred at the stage of image data sampling: the presampling MTF was inferior to the IP MTF due to the effect of the scattering and resultant spreading of the incidence laser beam and the emitted luminescence. The noise was characterized by means of digital Wiener spectrum using uniformly exposed noise data. Exposure ranges could be separated into different sections depending upon the noise sources, such as quantum mottle at low exposure and system structure noise at high exposure.

Comparison of two methods for accurate measurement of modulation transfer functions of screen-film systems.

The modulation transfer function (MTF) of a screen-film system can be measured by two methods, i.e., a slit method with Fourier transform on the line spread function and a square-wave response function (SWRF) method. However, it is still uncertain whether MTFs obtained by the two methods are identical. In this study, MTFs of relatively sharp and unsharp screen-film systems were measured by using the two methods. The slit method provided slightly greater MTF for the relatively sharp system than the SWRF method. However, MTFs of the unsharp system obtained with the two methods were comparable. Generally, the slit method tends to provide reliable results for unsharp systems, whereas the SWRF method is favorable for sharp systems. Accuracy and consistency of these measurements were examined by comparison of experimental and theoretical edge responses derived from the measured MTFs. However, the difference in edge responses obtained by the two methods was relatively small compared with the variation of the measured edge responses, and thus results were considered inconclusive as to whether either of the methods can provide more accurate MTFs. International interlaboratory comparison indicated that the variation in the measured MTFs at six different institutions was relatively large for both methods. However, the MTFs of two screen-film systems measured by the slit method appear to agree with those by the SWRF method within the variation expected from the interlaboratory comparison.

A simple method for determining the modulation transfer function in digital radiography.

The authors developed a simple method for determining the presampling modulation transfer function (MTF). which includes the unsharpness of the detector and the effect of the sampling aperture, in digital radiographic (DR) systems. With this method, the presampling MTF is determined by the Fourier transform of a ;finely sampled' line spread function (LSF) obtained with a slightly angulated slit in a single exposure. Since the effective sampling distance becomes much smaller than the original sampling distance of the DR system, the effect of aliasing on the MTF calculations can be eliminated. The authors applied this method to the measurement of the presampling MTF of a compound radiographic system and examined the directional dependence, the effect of exponential extrapolation, and the effect of different sampling distances. It is shown that the technique of multiple slit exposure and exponential extrapolation of the LSF tail, which has been commonly used in analog seven-film systems, can be employed in DR systems. The authors determined the glare fraction in order to estimate the component of low-frequency drop mainly due to ;glare'

Computerized analysis of interstitial infiltrates on chest radiographs: a new scheme based on geometric pattern features and Fourier analysis.

RATIONALE AND OBJECTIVES: Detection of interstitial infiltrates on chest radiographs is difficult and subjective. Therefore, we developed a computerized method to provide quantitative analysis of lung texture to increase diagnostic accuracy. METHODS: Two hundred chest radiographs--100 healthy and 100 abnormal with interstitial infiltrates--were digitized using a laser scanner. They were analyzed by an automated computerized scheme that uses a combination of two methods for detection of interstitial infiltrates: a lung texture analysis based on the Fourier transform and a geometric pattern feature analysis based on filtering techniques. RESULTS: The overall sensitivity and specificity of the computerized scheme were 92% and 90%, respectively. The scheme achieved a sensitivity of 80% in subtle cases (n = 15) and 88% in cases with localized interstitial disease (n = 26), whereas the specificity remained unchanged. There was good correlation between the computer output and the radiologists' severity rating. CONCLUSION: This enhanced computerized scheme exhibits high sensitivity and specificity with a large database.

Detectability of simulated interstitial pneumonia on chest radiographs: comparison between irradiation side sampling indirect flat-panel detector and computed radiography.

OBJECTIVE: To compare the detectability of simulated interstitial pneumonia on chest radiographs between an irradiation side sampling indirect flat-panel detector (ISS-FPD) and computed radiography (CR). METHODS: Simulated interstitial pneumonia findings (ground-glass opacity, reticular opacity and honeycomb lung) were superimposed on an anthropomorphic chest phantom. Chest radiographs were acquired under three exposure levels (4.0, 3.2 and 2.0 mAs) with an ISS-FPD and with CR. 5 thoracic radiologists evaluated 72 images for the presence or absence of a lesion over each of 6 areas. A total of 1296 observations were analysed in a receiver-operating characteristic analysis. A jackknife method was used for the statistical analysis. RESULTS: The areas under the curves (AUCs) for the detection of simulated honeycomb lung obtained with the ISS-FPD were significantly larger than those obtained with CR at all exposure conditions. For the detection of simulated ground-glass opacity and reticular opacity, there were no significant differences between the two systems. In addition, the AUCs for the detectability of simulated honeycomb lung obtained with the ISS-FPD at all exposure levels were significantly larger than those obtained with CR at 4 mAs. CONCLUSION: The ISS-FPD was superior to CR for the detection of simulated honeycomb lung. Provided that the chosen model is representative of interstitial pneumonia, the use of an ISS-FPD might reduce a patient's exposure dose during the detection of interstitial pneumonia. ADVANCES IN KNOWLEDGE: The ISS-FPD has shown its advantage compared with CR in the detection of honeycombing, one sign of interstitial pneumonia.

SU-E-I-59: Investigation of the Usefulness of a Standard Deviation and Mammary Gland Density as Indexes for Mammogram Classification.

PURPOSE: To investigate the usefulness of the standard deviation of pixel values in a whole mammary glands region and the percentage of a high- density mammary glands region to a whole mammary glands region as features for classification of mammograms into four categories based on the ACR BI-RADS breast composition. METHODS: We used 36 digital mediolateral oblique view mammograms (18 patients) approved by our IRB. These images were classified into the four categories of breast compositions by an experienced breast radiologist and the results of the classification were regarded as a gold standard. First, a whole mammary region in a breast was divided into two regions such as a high-density mammary glands region and a low/iso-density mammary glands region by using a threshold value that was obtained from the pixel values corresponding to a pectoral muscle region. Then the percentage of a high-density mammary glands region to a whole mammary glands region was calculated. In addition, as a new method, the standard deviation of pixel values in a whole mammary glands region was calculated as an index based on the intermingling of mammary glands and fats. Finally, all mammograms were classified by using the combination of the percentage of a high-density mammary glands region and the standard deviation of each image. RESULTS: The agreement rates of the classification between our proposed method and gold standard was 86% (31/36). This result signified that our method has the potential to classify mammograms. CONCLUSIONS: The combination of the standard deviation of pixel values in a whole mammary glands region and the percentage of a high-density mammary glands region to a whole mammary glands region was available as features to classify mammograms based on the ACR BI- RADS breast composition.

SU-E-I-21: Effect of Different Fluorescent Lights with Various Colors of a Reading Room on Chromaticity of LCD.

PURPOSE: To examine variation of chromaticity of LCD in different types of fluorescent lights in a reading room. METHODS: A color LCD (RX320, antiglare type, 450 cd/m2 , three-megapixel, Eizo Nanao), and a monochrome LCD (G31-S, anti glare type, 450 cd/m2 , three-megapixel, Eizo Nanao) were used in this study. The chromaticity in grayscale images with eighteen luminance levels were measured under five types of fluorescent lights with different color spectrums (Daylight: 6,700 K, Natural white: 5,000 K, White: 4,200 K, Warm white: 3,500 K, Light bulb: 3,000 K) by using a colorimeter (CS-200: KONICA MINOLTA). The chromaticity of LCDs was measured at various ambient lighting conditions (a dark room, 36, and 300 lux) and different types of fluorescent lights. RESULTS: The chromaticity of LCDs measured under ambient lights was changed from that measured in a dark room. The chromaticity of LCDs varied with different types of fluorescent lights. As illuminance of the room increased, variations in chromaticity at relatively lower luminance levels increased. The direction of changes in chromaticity shifted to the color for each fluorescent light. CONCLUSIONS: Fluorescent lights having different color spectra affect the chromaticity of LCDs.

SU-E-I-62: Investigation of Dominant Factors Affecting Fatigue in Image Reading of Radiologists.

PURPOSE: To investigate dominant factors affecting fatigue in image reading of radiologists. METHODS: Two kinds of fatigue were assessed in this study. One was fatigue in the central nervous system evaluated by the critical fusion frequency (CFF). The other was eye fatigue evaluated by a score determined from a questionnaire based on the oculomotor strain subscale from the Simulator Sickness Questionnaire (SSQ). When fatigue increases, the CFF and the SSQ score indicate low and high values, respectively. The fatigue of seventeen radiologists was assessed before and after their daily image reading. The reading times and the numbers of images were different among the assessments, and ranged about 1.5 - 5.0 hours and 1,000 - 12,000 images, respectively. The assessments of fatigue were repeated four times for each radiologist on different days. Finally, the measurements of the two kinds of fatigue were analyzed in terms of years of experience, age, sleeping time the previous night, ambient light conditions, reading time, and the numbers of interpreted images, series, and cases. RESULTS: The CFF and SSQ score after image reading were significantly lower and higher than those measured before image reading, respectively. Younger and less experienced radiologists indicated a higher level of fatigue than older and more experienced radiologists in both the CFF and the SSQ score. When radiologists interpreted clinical images for longer hours, the SSQ score tended to be higher. On the other hand, there was little incremental difference in the CFF among different lengths of reading time. No obvious differences were observed in the other items. CONCLUSIONS: Less experience with reading images, a younger age, and a longer reading time could be dominant factors affecting fatigue in image reading.

Quantitative Analysis of Trace Moisture in N(2) and NH(3) Gases with Dual-Cell Near-Infrared Diode Laser Absorption Spectroscopy.

This paper demonstrates our optical measurement system based on near-infrared tunable diode laser absorption spectrometry and reports the results of trace moisture determination in nitrogen and ammonia gases. A near-infrared InGaAsP distributed feedback diode laser operating at room temperature was employed as the optical source. We used a dual-cell detection strategy to cancel common mode noise from the diode laser and remove the effect of the residual moisture absorption in the beam path outside the sample cell. We also used this method to successfully eliminate the interfering absorption of matrix gas molecules such as NH(3). The detection limit of H(2)O absorption of 4 ppb in nitrogen and 12 ppb in ammonia was obtained using a single-pass absorption cell of only 92 cm in length and the average results of 10 scan measurements. This system has characteristics of both the high sensitivity and capability of in situ and real-time measurement.

Computer-aided diagnosis for detection of interstitial opacities on chest radiographs.

OBJECTIVE: Our objective was to evaluate the impact of a computer-aided diagnostic scheme on radiologists' interpretations of chest radiographs with interstitial opacities by performing an observer test using receiver operating characteristic (ROC) analysis. MATERIALS AND METHODS: Twenty chest radiographs with normal findings and 20 chest radiographs with abnormal findings were used. Each radiograph was divided into four quadrants. One hundred twenty-nine quadrants (80 normal and 49 abnormal quadrants) were used for testing because we excluded 31 equivocal quadrants. Sixteen independent observers (10 residents and six attending radiologists) participated in this study. The radiologists' performance without and with computer assistance, which indicated cases with normal and abnormal findings by various markers, was evaluated by ROC analysis. RESULTS: The diagnostic accuracy of the observers improved by a statistically significant magnitude when computer-aided diagnosis was used. Thus, the values for the area under the ROC curve obtained with and without the computer-aided diagnostic output were .970 and .948 (p = .0002), respectively, for all observers; .969 and .943 (p = .0006), respectively, for the residents' subgroup; and .972 and .960 (p = .162), respectively, for the attending radiologists' subgroup. The value for the area under the ROC curve for the computerized scheme by itself was .943. CONCLUSION: Our computer-aided diagnostic scheme can assist radiologists in the diagnosis or exclusion of interstitial disease on chest radiographs.

Quantitative analysis of geometric-pattern features of interstitial infiltrates in digital chest radiographs: preliminary results.

We are developing a computerized method for detection and characterization of interstitial diseases based on a quantitative analysis of geometric features of various infiltrate patterns in digital chest radiographs. In our approach, regions of interest (ROIs) with 128 x 128 matrix size (22.4 mm x 22.4 mm) are automatically selected, covering peripheral lung regions. Next, nodular and linear opacities, which are the basic components of interstitial infiltrates, are identified from two processed images obtained by use of a multiple-level thresholding technique and a line enhancement filter, respectively. Finally, the total area of nodular opacities and the total length of linear opacities in each ROI are determined as measures of geometric pattern features. We have applied this computer analysis to 72 ROIs with normal and abnormal patterns that were classified in advance by six chest radiologists. Preliminary results indicate that the distribution of measures of geometric-pattern features correlate well with radiologists' classification. These early results are encouraging, and further evaluation hopes to establish that this computerized method might prove useful to radiologists in their assessment of interstitial diseases.

Bir1/Cut17 moving from chromosome to spindle upon the loss of cohesion is required for condensation, spindle elongation and repair.

BACKGROUND: In mammals, proteins containing BIR domains (IAPs and survivin) are implicated in inhibiting apoptosis and sister chromatid separation. In the nematode, Bir1 is required for a proper localization of aurora kinase, which moves from the mitotic chromosome in metaphase to the spindle midzone in anaphase as a passenger. Fission yeast Bir1/Pbh1 is essential for normal mitosis. RESULTS: A temperature sensitive mutant cut17-275 exhibits the defect in condensation and spindle elongation at 36 degrees C, while securin is degraded. Gene cloning shows that the cut17+ gene is identical to bir1+/pbh1+. At 26 degrees C, cut17-275 is UV sensitive as the repair of DNA damage is severely compromised. Bir1/Cut17 is a nuclear protein in interphase, which is then required for recruiting condensin to the mitotic nucleus, and concentrates to form a discrete number of dots from prometaphase to metaphase. Once the chromatids are separated, Bir1/Cut17 no longer binds to kinetochores and instead moves to the middle of spindle. Chromatin immunoprecipitation suggested that Bir1/Cut17 associates with the outer repetitious centromere region in metaphase. Following the initiation of anaphase the protein switches from being a chromosomal protein to a spindle protein. This transit is stringently regulated by the state of sister chromatid cohesion proteins Mis4 and Rad21. Ark1, is an aurora kinase homologue whose mitotic distribution is identical to, and under the control of Bir1/Cut17. CONCLUSIONS: Bir1/Cut17 and Ark1 act as "passengers" but they may play a main role as a recruitment factor, essential for condensation, spindle elongation and DNA repair. Bir1/Cut17 should have roles both in mitotic and in interphase chromosome. The proper location of Ark1 requires Bir1/Cut17, and the mitotic localization of Bir1/Cut17 requires sister cohesion.

Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase.

Cohesin complex acts in the formation and maintenance of sister chromatid cohesion during and after S phase. Budding yeast Scc1p/Mcd1p, an essential subunit, is cleaved and dissociates from chromosomes in anaphase, leading to sister chromatid separation. Most cohesin in higher eukaryotes, in contrast, is dissociated from chromosomes well before anaphase. The universal role of cohesin during anaphase thus remains to be determined. We report here initial characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3, in fission yeast. They are essential for sister chromatid cohesion. Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and Psm3 but not with Psc3. Chromatin immunoprecipitation shows that cohesin subunits are enriched in broad centromere regions and that the level of centromere-associated Rad21 did not change from metaphase to anaphase, very different from budding yeast. In contrast, Rad21 containing similar cleavage sites to those of Scc1p/Mcd1p is cleaved specifically in anaphase. This cleavage is essential, although the amount of cleaved product is very small (<5%). Mis4, another sister chromatid cohesion protein, plays an essential role for loading Rad21 on chromatin. A simple model is presented to explain the specific behavior of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to be cleaved for normal anaphase.