Acetylcholine-induced coronary vasoconstriction and negative inotropy in the neonatal pig heart.

We investigated the influence of exogenously administered acetylcholine, nitric oxide, ADP, ATP, bradykinin, and substance P on coronary vascular tone in isolated, neonatal pig hearts (less than or equal to 4 d). Paced (180 bpm), isovolumically beating hearts underwent retrograde aortic perfusion, with an erythrocyte-enriched solution (hematocrit 0.15-0.20) at constant coronary flow (approximately 2.5 mL/min/g) corresponding to a perfusion pressure of approximately 60 mm Hg. Agonists were injected into the aortic root, and the peak change in coronary perfusion pressure from baseline and left ventricular pressure development were assessed. Nitric oxide (3 microL), ADP (30 nmol), ATP (30 nmol), bradykinin (125 ng), and substance P (50 ng) decreased the perfusion pressure (vasodilation) by 16.9 +/- 1.2, 25.3 +/- 4.4, 18.3 +/- 1.2, 18.9 +/- 1.4, and 7.1 +/- 1.6 mm Hg, respectively. Acetylcholine (0.5 and 1.0 nmol) produced a modest decrease in perfusion pressure (vasodilatation) of 4.2 +/- 0.8 and 3.8 +/- 0.5 mm Hg, respectively, whereas acetylcholine (5, 20, and 100 nmol) increased the perfusion pressure (vasoconstriction) by 16.7 +/- 2.7, 48.2 +/- 8.2, and 85.3 +/- 15.1 mm Hg, respectively. Acetylcholine also decreased left ventricular peak systolic pressure from 108.7 +/- 5.0 to 69.2 +/- 4.6, 56.3 +/- 6.1, and 48.2 +/- 6.4 mm Hg, for the 5, 20, and 100 nmol doses, respectively. Responses to acetylcholine were abolished by atropine (50 nmol). In a separate group of hearts, indomethacin (10(-6) M) reduced the peak change in perfusion pressure for the 5, 20, and 100 nmol doses of acetylcholine by 87%, 66%, and 48%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Random-zero sphygmomanometer, Rose's tape, and the accuracy of the blood pressure measurements in children.

In the Cardiovascular Risk in Young Finns project, blood pressure (BP) was measured in 3549 randomly selected children aged 6-18 y in 1980, and 2887 and 2500 of the same individuals in 1983 and 1986, respectively. An ordinary mercury sphygmomanometer (OMS) was used in the first two surveys and a random-zero sphygmomanometer (RZS) in the third survey. Systolic and diastolic BP were lower when measured with an RZS than with an OMS and the shape of the age-related BP curve obtained with an RZS was significantly different from that obtained with an OMS, because low BP values were apparently measured more accurately with the former. Use of the RZS did not affect the distribution of the BP values. Rose's tape readings were used to evaluate the effect of training and to control the accuracy of the BP measurements. According to Rose's tape readings, Korotkoff's 4th phase BP was more difficult to measure accurately than 5th phase (p = 0.002). The mean values for the differences between the correct and actual BP readings on the Rose's tape were -1.2 mm Hg (SD 2.1) for systolic BP, 8.3 mm Hg (SD 13.6) for diastolic Korotkoff's 4th phase BP, and 1.2 mm Hg (SD 7.0) for diastolic Korotkoff's 5th phase BP, with a negative value indicating that the BP phase was measured lower than the correct value. Terminal digit preference was present in each survey to such an extent that it could have hampered the reaching of reliable conclusions from the data obtained with an OMS. The bias caused by terminal digit preference was obviated by the RZS.(ABSTRACT TRUNCATED AT 250 WORDS)


Attention in visual search: multiple search classes.

Data from visual-search tasks are typically interpreted to mean that searching for targets defined by feature differences does not require attention and thus can be performed in parallel, whereas searching for other targets requires serial allocation of attention. The question addressed here was whether a parallel-serial dichotomy would be obtained if data were collected using a variety of targets representing each of several kinds of defining features. Data analyses included several computations in addition to search rate: (1) target-absent to target-present slope ratios; (2) two separate data transformations to control for errors; (3) minimum reaction time; and (4) slopes of standard deviation as a function of set size. Some targets showed strongly parallel or strongly serial search, but there was evidence for several intermediate search classes. Sometimes, for a given target-distractor pair, the results depended strongly on which character was the target and which was the distractor. Implications from theories of visual search are discussed.


Auditory apparent motion in the free field: the effects of stimulus duration and separation.

The effects of stimulus duration and spatial separation on the illusion of apparent motion in the auditory modality were examined. Two narrow-band noise sources (40 dB, A-weighted) were presented through speakers separated in space by 2.5 degrees, 5 degrees, or 10 degrees, centered about the subject's midline. The duration of each stimulus was 5, 10, or 50 msec. On each trial, the sound pair was temporally separated by 1 of 10 interstimulus onset intervals (ISOIs): 0, 2, 4, 6, 8, 10, 15, 20, 50, or 70 msec. Five subjects were tested in nine trial block; each block represented a particular spatial-separation-duration combination. Within a trial block, each ISOI was presented 30 times each, in random order. Subjects were instructed to listen to the stimulus sequence and classify their perception of the sound into one of five categories: single sound, simultaneous sounds, continuous motion, broken motion, or successive sounds. Each subject was also required to identify the location of the first-occurring stimulus (left or right). The percentage of continuous-motion responses was significantly affected by the ISOI [F(9,36) = 5.67, p less than .001], the duration x ISOI interaction [F(18,72) = 3.54, p less than .0001], and the separation x duration x ISOI interaction [F(36,144) = 1.51, p less than .05]. The results indicate that a minimum duration is required for the perception of auditory apparent motion. Little or no motion was reported at durations of 10 msec or less. At a duration of 50 msec, motion was reported most often for ISOIs of 20-50 msec.(ABSTRACT TRUNCATED AT 250 WORDS)


Adaptation to auditory motion in the horizontal plane: effect of prior exposure to motion on motion detectability.

Thresholds for auditory motion detectability were measured in a darkened anechoic chamber while subjects were adapted to horizontally moving sound sources of various velocities. All stimuli were 500-Hz lowpass noises presented at a level of 55 dBA. The threshold measure employed was the minimum audible movement angle (MAMA)--that is, the minimum angle a horizontally moving sound must traverse to be just discriminable from a stationary sound. In an adaptive, two-interval forced-choice procedure, trials occurred every 2-5 sec (Experiment 1) or every 10-12 sec (Experiment 2). Intertrial time was "filled" with exposure to the adaptor--a stimulus that repeatedly traversed the subject's front hemifield at ear level (distance: 1.7 m) at a constant velocity (-150 degrees/sec to +150 degrees/sec) during a run. Average MAMAs in the control condition, in which the adaptor was stationary (0 degrees/sec,) were 2.4 degrees (Experiment 1) and 3.0 degrees (Experiment 2). Three out of 4 subjects in each experiment showed significantly elevated MAMAs (by up to 60%), with some adaptors relative to the control condition. However, there were large intersubject differences in the shape of the MAMA versus adaptor velocity functions. This loss of sensitivity to motion that most subjects show after exposure to moving signals is probably one component underlying the auditory motion aftereffect (Grantham, 1989), in which judgments of the direction of moving sounds are biased in the direction opposite to that of a previously presented adaptor.


The role of symmetry in determining perceived centers within shapes.

This study was designed to assess the effects of symmetry and plane of presentation on the determination of the perceptual center of flat figures. Experiment 1 demonstrates the existence of effects in improving center determination, both in the number of sides of the shape and in rotational and reflective symmetry (confounded in the experiment). Experiment 2 shows that the presentation plane has no effect on center determination. In Experiment 3, we divide the effects of the two symmetry types, showing that rotational symmetry alone is as effective as the presence of both symmetry types--that is, the presence of symmetry axes is not very useful in finding perceived centers.


Orientation-contingent color aftereffects mediated by subjective transparent structures.

We examined whether the orientation-contingent color aftereffect (the McCollough effect) could be mediated by subjective horizontal and vertical structure induced by the perception of transparency. In our experiments, red vertical bars and green horizontal bars were alternated as an adapting stimulus. After adaptation, subjects (n = 6) were asked to adjust the green and red saturation of a test pattern until they obtained a neutral gray. Horizontal and vertical stripes were combined in the test pattern in three different ways: (1) overlapping with a luminance combination that gave rise to a perception of transparent overlays of horizontal and vertical stripes (valid transparency condition), (2) overlapping with luminance combinations that did not induce a perception of transparency (invalid transparency condition) and that appeared more as a patchwork of checks, and (3) presented in adjacent, nonoverlapping areas. Our results showed that the McCollough effect was significantly greater in the valid transparency condition than in the invalid transparency conditions. The effect in the valid transparency condition was nevertheless less strong than was the effect seen with the standard test stimulus made up of nonoverlapping vertical and horizontal stripes. Our results suggest that the McCollough effect can be mediated by the subjective spatial organization (inner representation of vertical and horizontal stripes) that accompanies the perception of transparency in our stimulus.


Saccades to targets in three-dimensional space: dependence of saccadic latency on target location.

The latency of saccadic movements to targets appearing at various positions in three-dimensional visual space was measured in four experiments. The first experiment confirmed that latencies of saccades to visual targets are greater in the lower visual field and showed that the increase is not influenced by the vertical starting position of the eye in the orbit, nor by a time gap between the fixation offset and the target onset. A hypothesis that this visual field difference was caused by a link between downward saccades and convergence movements was tested by recording saccade latencies when the targets were in a different depth plane from that of the original fixation. We did not find any direct support for the vergence involvement hypothesis, although the lower/upper visual field effect was shown to decrease consistently in monocular viewing. It was also shown that saccades to targets positioned in a different depth plane have longer latencies. In a final experiment, the visual field effect was shown to depend on the egocentric rather than the gravitational vertical.


Allocation of visual attention in younger and older adults.

Younger and older adults were compared in three experiments, using procedures that had been shown to affect the spread of visual attention. The attentional effects found in previous experiments were replicated. A broader focus of attention speeded responses to peripheral targets. In addition, two established findings concerning aging were replicated: Responses were slower in older than in younger adults, and, in certain conditions, they slowed more rapidly as target eccentricity increased. No interactions of age effects with attentional manipulations were found. The results of all three experiments were consistent with the interpretation that younger and older adults do not differ in the allocation of attention.


Suppression of optokinesis by a stabilized target: effects of instruction and stimulus frequency.

Subjects viewed a foveally stabilized target presented against a background field of dots moving sinusoidally. Several different modes of viewing the target were used (subjects were instructed to gaze, look, or hold), and the frequency of sinusoidal field motion was varied from 1/32 to 2 Hz. In line with previous findings, the presence of a stabilized target resulted in substantial suppression of optokinesis. The characteristics of this suppression (gain and phase of slow residual eye movements) were dependent on both the mode of viewing the target and the frequency of field motion. When subjects used an imaginary target, little suppression occurred. These findings provide an overall profile of dynamic characteristics of mechanisms involved in the suppression of optokinesis. They support the view that this suppression is significantly determined by the presence of a target against a moving background (even without retinal slip), and by the mode of attending to the target.


The effects of complexity on the perception of vibrotactile patterns presented to separate fingers.

Pairs of vibrotactile patterns were presented to subjects' left middle and index fingerpads (unilateral presentation) or left and right index fingerpads (bilateral presentation), using two Optacon arrays. A set of simple (one-line) patterns and a set of complex (two-line) patterns were constructed so that they were equally identifiable when presented individually. In Experiment 1, discrimination performance was lower for two-line patterns than it was for one-line patterns, and it was lower for unilateral presentation than it was for bilateral presentation. Communality, the number of lines that two patterns share in common, was a major factor in reducing discrimination performance for two-line patterns. Subjects' abilities to identify one member of the pair of patterns were measured in Experiment 2. There were no significant differences in performance between pattern sets or type of presentation when subjects attended to a single pattern. However, when subjects were required to attend to both patterns, identification performance was lower for two-line patterns than it was for one-line patterns, and it was lower for unilateral presentation than it was for bilateral presentation. The results suggest that there are limited attentional resources for processing vibrotactile patterns and that more resources are available bilaterally than are available unilaterally.


Spatial attention in Ponzo-like patterns.

A variant of Ponzo's pattern was devised by drawing converging oblique lines at the ends of each of two horizontal lines that were located directly one above the other. Pairs of converging or diverging fins (either between or outside the horizontal shafts) were systematically removed. The results showed that, as predicted by integrative field theory, oblique lines between the shafts produced more distortion than did oblique lines outside the shafts. Also, it was shown that the attentive field construct in integrative field theory was crucial for predicting patterns of means and variances in this experiment. The modified Ponzo effect was shown to have much in common with the classical Müller-Lyer illusion. The subtle differences between the two were explained by proposing that the size of the optimum attentive field was larger in the Ponzo tasks than in the Müller-Lyer tasks because expansion fins are found on both shafts in the Ponzo-like figure.


Apparent duration and spatial structure.

In three experiments, we investigated the relative perceived duration of a full bandwidth image and a set of high- and lowpass filtered images of a scene, briefly presented on a visual display unit. In Experiment 1, the various images were compared with each other, using a paired comparison method. All images were presented for 40 msec, and observers were asked to judge which of each pair of images had the longest duration. The results showed that images containing a wide spatial frequency bandwidth were judged to be of longer duration than were images of a narrower bandwidth, regardless of whether the latter were high- or lowpass filtered. In Experiment 2, a 40-msec presentation of each of the images was compared with a presentation of a probe that was 20, 40, 60, or 80 msec in duration. Observers again judged which of each pair of images had the longest duration. The results were very similar to those of Experiment 1, with wide bandwidth images being judged to be of longer duration than were narrow bandwidth images. In Experiment 3, instead of comparing the various filtered versions of the image with each other, we attempted to obtain a direct measure of perceived duration by comparing a flashing LED to a 40-msec flash of a subset of the images used in the previous experiments. The observers' task was to adjust the duration of the LED flash to match the perceived duration of each image.(ABSTRACT TRUNCATED AT 250 WORDS)


[Growth rate of abdominal aortic aneurysms as measured by Computerized tomography].

To evaluate the growth rate of abdominal aortic aneurysms, sequential computed tomography (CT) examinations were performed on 125 nonoperated patients (M/F 101/24, mean age 67 +/- 8 years, 129 lesions) at intervals of at least 6 months. Growth rates of aneurysms were obtained by subtracting the first from the last diameter and adjusting for examination interval. Mean growth rate was 0.28 +/- 0.26 cm/year. Aneurysms with an initial diameter exceeding 4 or 5 cm showed significantly faster growth than smaller aneurysms. There was no significant correlation between growth rate and atherosclerogenic factors, but systolic blood pressure was significantly higher in patients whose aneurysms ruptured. Our study showed quantitatively that aneurysms with a large diameter have a rapid growth rate, the growth rate of infrarenal aneurysms is significantly faster than that of suprarenal aneurysms, and blood pressure control is important to prevent rupture.


[Clinical evaluation of stent placement for tracheal and bronchial stenosis].

Expandable metallic stents were successfully introduced in 7 patients, including 4 with left main bronchial stenosis caused by bronchopulmonary tuberculosis, 2 with main bronchial stenosis caused by lung cancer and one with tracheal stenosis caused by adenoid cystic carcinoma. The length of stenosis was 1.5-5 cm. The stents were 1.5-2.5 cm long with barbs, and their full expanded diameter was 1.5 cm. Balloon dilatation was performed before stenting in all cases. The stents were inserted by using a 10-12 Fr catheter. In all patients except the one with tracheal stenosis, stents were introduced under local anesthesia without any difficulties. No migration of stents occurred. After stent placement, there were no respiratory difficulties, and radionuclide lung perfusion scan and chest radiographic findings such as lung atelectasis showed marked improvement in three cases. Combined therapy of stent placement and bronchial arterial infusion chemotherapy showed marked effectiveness in one case with lung cancer. Expandable metallic stents were very useful in eliminating tracheobronchial stenosis symptoms.


[Magnetic resonance (MR) imaging of chordoma and chondroma in the skull base--differential diagnosis by IR sequence].

Differential diagnosis of chordoma and chondroma in the skull base is sometimes difficult. We retrospectively reviewed the MR images of 14 patients with skull base tumors (nine chordomas, four chondromas and one chondrosarcoma). MR imaging was performed with a 0.5 Tesla system (Picker International). Inversion recovery (IR) (2500-2100/600-500/40), T1-weighted spin echo (SE) (800-600/40), and T2-weighted SE (2500-1800/120) images were obtained. On IR images, seven of eight chordomas showed heterogeneous low signal intensity, and one chordoma and all chondromas showed markedly low signal intensity similar to that of CSF. Calcified or ossified portions of the chondromas were demonstrated as areas of moderately low intensity on IR images. Chondrosarcoma showed moderately low intensity similar to that of chordoma. T1-weighted SE images of chordoma and chondroma showed no difference in signal intensity. On T2-weighed SE images, six of nine chordomas and all chondromas showed markedly high signal intensity. Three chordomas and one chondrosarcoma showed moderately high signal intensity. In the diagnosis of skull base tumors, the IR sequence seems to be useful for differentiating chondroma from chordoma.


[Angiographic features of alveolar soft-part sarcoma].

The angiographic findings were analyzed in five patients with alveolar soft-part sarcoma. The angiographic appearance of the tumors was the same in all five cases: hypervascularity of the tumor including encasement, dilatation, tortuosity and displacement of feeding arteries, remarkable tumor stain and early venous filling. These angiographic findings are considered to be characteristic of alveolar soft-part sarcoma in the differential diagnosis of hypervascular soft tissue mass.


[Computerized tomography in pulmonary infarction].

We performed a retrospective study of CT findings in 14 patients with 61 lesions of pulmonary infarction diagnosed clinically and radiographically. All cases except one had multiple pleural-based parenchymal lesions, and in five cases the lesions were bilateral. The site of pulmonary infarction was the right lower lobe in 24 lesions, left lower lobe in 19 lesions, right upper lobe and left upper lobe in seven lesions each, and right middle lobe in four lesions. Fifty-nine infarctions (excluding two segmental lesions) were divided into two groups according to the size of the lesions: a group of lesions 2 cm or more in size and a group less than 2 cm. Nineteen lesions in eight patients were in the larger group and 40 lesions in 12 patients in the smaller group. The typical CT findings of larger infarctions were pleural based parenchymal density with truncated apex, centrally directed linear shadow and inside low attenuation area indicating viable lung. Pleural thickening and effusion were frequently seen. No air bronchograms were seen in these subsegmental lesions. Smaller lesions were believed to indicate infarction of a single secondary pulmonary lobule, considering their size and shape. CT scan was able to detect these small lesions (single lobular infarctions) more frequently than chest radiography. One segmental lesion with air bronchogram was thought to represent atelectasis and the other one to represent pulmonary hemorrhage and edema. CT examination is useful for the detection and diagnosis of pulmonary infarction.


[An experimental and clinical study of zonography of the lung by means of Fuji computed radiography].

Tomography was conducted for a basic study using a model of the bronchi, pulmonary arteries and veins. When the exposure angle of a tube was changed to 5, 10, 25, and 50 degrees, the width of tomograms became about 10, 4, 2, and 1 cm. When tomograms of exposure angles of 50 degrees and 10 degrees were compared, the former showed fragmentary bronchi, pulmonary arteries and veins, and the latter showed bronchi, pulmonary arteries and veins branching up, down and laterally on one image in one unit and over a wide area. Similar results were obtained in 10 cases of normal volunteer. When 25 cases of lung cancer at the hilum were studied clinically with respect to bronchial lesions, the tomograms with an exposure angle of 10 degrees could point out more lesions than the tomograms with an exposure angle of 50 degrees. In 13 cases (40 lesions) which were compared with the findings of bronchoscopy, the accuracy was only 25% in tomograms with an exposure angle of 50 degrees, and 65% in tomograms with an exposure angle of 10 degrees. These results indicate that tomograms with an exposure angle of 10 degrees are useful for analysis of the bronchi, pulmonary arteries and veins at the hilum of the lung.


[Biliary endoprosthesis of malignant biliary obstruction using expandable metallic stent--preliminary clinical evaluation].

Thirty-eight patients with malignant biliary obstruction were treated with expandable metallic stent (EMS). Successful insertion of stents was attained in all cases, and in 34 of 38 patients, the stents remained patent and sufficiently expanded, and led to the removal of external drainage catheter. No serious complications occurred. In two cases, stents were deformed in shape, associated with no side effects. In follow-up, eleven patients developed recurrent jaundice due to tumor ingrowth between the wires and the tumor grew up along the initially placed stents. In six patients, additional stents were installed inside the initially placed stents, in whom the additional stents were placed successfully and remained patent thereafter. The expandable metallic stents were superior to the conventional tube stents, but there were some problems in our limited experience: stents deformity, slipping migration, fracture of stents and rapid obstruction. Further investigation is under way to resolve these problems. Long-term trials are required before established routine use of EMS. The expandable metallic stent was, however, expected to offer a new alternative in the management of malignant biliary obstructions and to afford long-term patency of affected biliary tracts.


[Temperature distribution and geometry of the electrodes in RF interstitial hyperthermia using circular and interstitial electrodes].

To evaluate the feasibility of clinical application of a newly developed interstitial hyperthermia system, which consists of an 8 MHz radiofrequency generator, interstitial needle electrodes, and a superficial circular electrode, we conducted preclinical experiments using an agar phantom and VX-2 carcinoma in the rabbit. In the experiment with an agar phantom, four 4 cm needle electrodes were placed in a square array at intervals of 1.0, 1.5, and 2.0 cm. Thermography demonstrated homogeneous temperature distribution at electrode intervals of 1.0 and 1.5 cm, but hot spots around the electrodes at an interval of 2.0 cm. When electrode deviation was less than 8 degrees from the parallel plane, no temperature deviation was observed. Using two 2 cm electrodes and two 4 cm electrodes in square array, thermography demonstrated a homogeneous temperature distribution in the area surrounded by the electrodes. Even if the electrodes were located at the periphery of the agar phantom, a homogeneous temperature distribution was obtained in the area surrounded by the electrodes. Using four 4 cm electrodes at intervals of 1.5 cm in VX-2 carcinoma in the rabbit, ideal heating was obtained: 42 degrees C at the periphery of the tumor and 43 degrees C at the center. These data suggest that the newly developed interstitial hyperthermia apparatus provides homogeneous heat distribution at electrode intervals of 1.5 cm or less and can be used in a Phase I study for deep-seated or superficial tumors.


[Single photon emission computed tomography by using fan beam collimator].

A multislice fan beam collimator which has parallel collimation along the cephalic-caudal axis of a patient and converging collimation within planes that are perpendicular to that axis was designed for a SPECT system with a rotating scintillation camera, and it was constructed by the lead casting method which was developed in recent years. A reconstruction algorithm for fan beam SPECT was formed originally by combining the reconstruction algorithm of the parallel beam SPECT with that of the fan beam X-ray CT. The algorithm for fan beam SPECT was confirmed by means of computer simulation and a head phantom filled with diluted radionuclide. Not only 99mTc but also 131I was used as a radionuclide. A SPECT image with the fan beam collimator was compared with that of a parallel hole, low energy, high resolution collimator which was routinely used for clinical and research SPECT studies. Both system resolution and sensitivity of the fan beam collimator were approximately 20% better than those of the parallel hole collimator. Comparing SPECT images obtained from fan beam collimator with those of parallel hole collimator, the SPECT images using fan beam collimator had far better resolution. A fan beam collimator is a useful implement for the SPECT study.


[Evaluation of thermal damage after hyperthermia on murine experimental tumor by 31P-NMR spectroscopy--correlation between ATP and growth curve].

The possibility of using 31P-NMR spectroscopy (31P-MRS) to estimate the effect of hyperthermic treatment on mouse FM3A tumor was investigated. 1 x 10(6) cells, suspended in saline, were subcutaneously inoculated to the right thigh of C3H mice. For hyperthermic treatment, the tumor-bearing leg was heated by immersing it in a water bath at 44 degrees C for 10, 20 or 30 min. The signal intensities of ATP and Pi of the tumor were measured utilizing the 31P-MRS technique to calculate the ATP/Pi ratio. Immediately after heating, the ATP/Pi ratio decreased markedly. Eighteen hours after hyperthermic treatment, the ratio recovered but was still smaller than the control value, then became almost constant by 24 hours after heating. The ATP/Pi ratio at 24 hours after heating decreased with increased length of heating and was inversely related to tumor regrowth after hyperthermic treatment. We concluded that non-invasive monitoring with 31P-MRS might provide a good indication of the effect of hyperthermic treatment.


[New puncture technique for TIPSS (transjugular intrahepatic portosystemic stent shunt)].

To establish a safer puncture method for TIPSS (Transjugular Intrahepatic Portosystemic Stent Shunt), we developed a new percutaneous puncture technique to penetrate portal and hepatic vein trans-hepatically under ultrasonography (US technique). Experimentally we compared this technique with conventional transjugular technique puncturing portal vein through hepatic vein using Ross needle (Ross Needle technique). Success rate is 100% in US technique and 50% in Ross needle technique. Seven complication including intraperitoneal bleeding occurred in Ross needle technique, but no complication in US technique. Conclusively, US technique is a safer and more reliable method than that using Ross needle in the puncture method for TIPSS.


[Biliary enhanced MR imaging by Gd-DTPA].

Biliary enhanced MRI (BEMRI) by Gd-DTPA via PTCD and/or PTGBD tube for obstructive jaundice was performed in 8 patients. In all cases, biliary tract was clearly visualised as high signal intensity on T1 weighted images. On same images, primary lesion such as common bile duct cancer was also visualised as well as portal system. In addition, MR angiography (MRA) by 2D-time of flight method was performed. MRA with BEMRI shows portal encasement on the same image as biliary tract obstruction. It suggests MRA with BEMRI may replace the other modality for obstructive jaundice.


[Experimental study on the usefulness of the autoperfusion balloon catheter in maintaining the blood supply to the distal organs].

Autoperfusion Balloon Catheters (APBC) were inserted into the renal arteries of seven dogs for about two and a half hours. Abdominal aortography showed renal arterial flow of the kidney obstructed by APBC. Macroscopic and microscopic examination revealed no significant changes in the resected specimen. It is concluded that APBC is useful in cases of proximal arterial occlusion by maintaining the blood supply to the distal organs for relatively short periods and that it can be used for the treatment of rupture of arteries which feed critical organs.


[Radiosensitizing effect of a 2-nitroimidazole hydroxamate (KIN-804) to murine tumors].

We studied the toxicity, pharmacokinesis and radiosensitizing effect of a newly developed 2-nitroimidazole-1-methylacetohydroxamate (KIN-804) in C3H/He mice bearing SCC-VII tumor. They were compared with misonidazole. LD50/7 of KIN-804 and misonidazole were 3200 and 2000 mg/kg. The concentration of KIN-804 in the brain and sciatic nerve was at a very low level and its clearance from the sciatic nerve was rapid. Enhancement ratios calculated using the growth delay method were 1.50 for KIN-804 and 1.36 for misonidazole respectively when they were administered by intraperitoneal injection with a dose of 100 mg/kg. KIN-804 was considered to be a promising radiosensitizer because it obtained less toxicity and a higher radiosensitizing activity than misonidazole.


[Effect of growth rate of monolayer cells on survival after fractionated irradiation].

This study was performed to determine the effect of the growth rate of monolayer cells on survival following fractionated irradiation. HeLa and RMUG cells that had different radiosensitivities and growth rates (Do value: 2.3 vs 1.5 Gy, doubling time: 17 vs 46 hours) were irradiated with 2 Gy every day. The fractions surviving after fractionated irradiation were compared with those given single doses. The dose modifying factor for fractionated irradiation was larger in RMUG than HeLa: 1.7 and 1.2, respectively. Two clones from ADGU cells that had the same radiosensitivity but different growth rates were also given fractionated irradiation, but there was no difference in surviving fractions. When recovery following two split doses was determined in each type of cell, the results of the fractions surviving after fractionated irradiation were correlated only with recovery between split doses. These suggest that the growth rates of monolayer cells may not modify the fractions surviving after fractionated irradiation, and the monolayer cell system is not suitable for determining the effect of growth rates on survival following fractionated irradiation.


[Analysis on combined effect of X-rays with 5-FU on rat subcutaneous gliomas].

In a series of experiments on the combined use of radio- and chemotherapy for malignant glioma, X-rays combined with ACNU or 5-FU treatment caused a supra-additive effect on multicellular spheroids in vitro. In the present experiment, the effect of X-rays combined with 5-FU treatment on subcutaneously transplanted rat gliomas of RGC-6 cells was analyzed. The dose-survival curve for X-rays given to tumors in air-breathing rats was biphasic with a terminal slope (D0 = 4.3 Gy) that was parallel to that for tumors in previously killed rats. The hypoxic cell fraction thus obtained from the ratio of the surviving fraction in two parallel curves at 20 Gy was about 7% in the subcutaneous tumors in air-breathing rats. X-ray-induced, potentially lethal cellular damage recovered within 8 hours in these tumors. The surviving fraction of cells in the tumors decreased to a minimum at 4-6 hours after a 5-FU injection, but increased thereafter. A biphasic dose-response curve for 5-FU was also obtained for cells in these tumors, indicating the presence of 5-FU resistant cells. The effect of X-irradiation given at about 8 hours after a 5-FU injection was greater than the additive effect of both agents acting independently. This was true when an X-ray dose of more than 5 Gy was given.


[Evaluation of US contrast medium (LEVOVIST) to experimental liver cancer].

Enhancement of liver cancer on ultrasonography (US) by injection of new contrast medium (LEVOVIST) was performed in rats with 3'-methyl-4-dimethylaminoazobenzene-induced hepatic carcinoma. Echosignals of cancer nodules increased remarkably after the intrahepatic arterial injection of LEVOVIST (200, 300 mg/ml), and contrast enhancement was observed for at least 15 minutes. Furthermore, US after the intrahepatic arterial injection of LEVOVIST (200 mg/ml) can visualize small nodule, which was not recognizable on plain US. As a result of increased echosignals of normal liver parenchyma after the intra-portal injection of LEVOVIST (300 mg/ml), the cancer nodules were demonstrated as hypoechoic. These results indicate that this new contrast medium for sonography is effective in the diagnosis of liver cancer.


[Gd-DTPA-enhanced MR imaging of the normal facial nerve].

We performed a prospective imaging study of the normal facial nerve within the temporal bone before and after injection of Gd-DTPA. The study included 29 patients using a 1.5T superconducting unit and 40 nerves (right: 21; left: 19) were available for analysis. There was no enhancement of the facial nerve within the internal auditory canal in the entire series. However, the enhancement at the labyrinthine segment was observed in one nerve (3%); at the geniculate ganglion in seven (18%); at the tympanic segment in 18 (45%) and at the mastoid segment in 28 nerves (70%). Our results indicate that enhancement of the facial nerve in normal subjects is not a rare condition.


[The study of ultrasonographic diagnosis on breast diseases].

15 sonomammographic findings of 341 histologically proved breast masses were reviewed to improve and objectify the current diagnostic criteria of sonomammography. The author proposed the renewed plan of the diagnostic criteria and the judgement method of benign or malignant breast masses on an electronic linear scanner, offering the author's original diagnostic concept particularly about the shape and the internal echo. This diagnostic approach demonstrated a sensitivity of 93.2%, specificity of 85.9%, and accuracy of 89.7%, which sufficiently showed to raise differential diagnostic capability for breast masses.


[Raise up 99mTc-HMPAO brain SPECT for detecting the changes in cerebral perfusion pressure].

99mTc-HMPAO crossed the blood brain barrier instantly in proportional to cerebral blood flow. Raise up stress and supine resting 99mTc-HMPAO with brain SPECT was devised for detecting abnormal response in the changes of cerebral perfusion pressure. Asymmetric ratio in the middle cerebral artery area in patients with internal carotid artery occlusion (n = 5) was changed significantly from 0.82 +/- 0.06 to 0.90 +/- 0.06 and that of normal (n = 5) was not changed from 0.94 +/- 0.03 to 0.95 +/- 0.01. Raise up 99mTc-HMPAO brain SPECT enhanced the detectability in abnormality of regional cerebral blood flow and visualized the dys-autoregulated area during blood pressure falls.


[Development of a gel phantom for dose distributions by MR imager].

We reported a method of determining spatial dose distributions in tissue-equivalent phantoms using nuclear magnetic resonance imaging. To make a gel phantom, a cross-linked dextran gel (Sephadex G-200) was used as fixation agent of a ferrous sulphate solution. Although high quality images are obtained, the cost is very expensive to make the gel. We developed a new gel which is combined with an extra water absorbent polymer (Sumikagel N-100) and Sephadex G-200. The gel permits the determination of dose distributions for three dimensional treatment planning.


[Evaluation of aortic distensibility using cine MR imaging].

Regional aortic distensibility was measured noninvasively using cine MR imaging to determine whether it correlates with aging or risk of coronary artery disease (CAD). Twenty-two patients with CAD confirmed by angiography and 33 without CAD underwent cine MR imaging. Thirty-two sequential images were obtained in one cardiac cycle. The cross sectional area of the descending thoracic aorta was measured on both diastolic (A) and systolic (A') images. Aortic distensibility (A.D.) was calculated from the following equation: A.D. = (A'-A)/A/delta P. (delta P represents pulse pressure). Results of a simple regression analysis showed that decreased regional aortic distensibility was significantly correlated with the age of the patients (r = -0.762, p less than 0.005). In addition, the parameter was lower in patients with CAD than those without CAD (p less than 0.05). In conclusion, regional aortic distensibility derived from cine MR imaging is a useful parameter to evaluate not only aging but also pathological atherosclerosis of the aorta. In addition, this parameter might have some potential to evaluate the prediction of CAD in patients with normal serum cholesterol.


[Complications of arterial infusion of CDDP in the treatment of malignant neoplasms].

Arterial infusion of cis-diamminedichloroplatinum (CDDP) is an effective therapy for malignant neoplasms. Severe complications after this therapy in 298 patients with malignant tumors were reviewed in our institute from March 1982 to August 1989. Arteries infused with CDDP and the number of infusions were as follows: bronchial artery (a.) 277, hepatic a. 153, internal iliac a. 51, femoral or popliteal a. 15, lumbar a. 6, pulmonary a. 5, esophageal a. 3 and intercostal a. 3. Eight cases of severe complications were encountered: 3 of esophageal ulcer, 3 of spinal paralysis, and one each of mediastinitis and toe necrosis. The arteries responsible for complications were the bronchial a. in 3 cases of esophageal ulcer and 2 of spinal paralysis, intercostal a. in one of spinal paralysis, esophageal a. in one of mediastinitis and popliteal a. in toe necrosis. Complication rates were 2.7% (8/298 cases) and 1.5% (8/518 infusions). Angiotensin II was infused in 6 of 8 of the cases with complications. The complication rate was 2.9% (6/209 infusions) in the group with CDDP and angiotensin II and 0.6% (2/309 infusions) in the group with CDDP only. Arterial infusion of CDDP, especially combined with angiotensin II, should be done in consideration of the arteries responsible for such complications.


[Phosphorus 31-magnetic resonance spectroscopic studies of animal liver after extracorporeal shock wave lithotripsy].

The technique of magnetic resonance spectroscopy has been developed, and the study of high-energy phosphate metabolites in the liver using phosphorus 31 magnetic resonance spectroscopy (31P-MRS) has been reported in humans and animals, but few studies have used 31P-MRS for the evaluation of extracorporeal shock wave lithotripsy (ESWL) of the liver. In this study, 31P-MRS was used to evaluate the metabolic changes in hamster liver after ESWL and histological correlation was performed. Syrian golden hamsters were anesthetized and shock waves were irradiated to the left side of the liver. Hamsters were irradiated by LITHOSTAR-PLUS (SIEMENS) at a voltage of 19 KV. 31P-MRS was studied by JNM-GSX model 270 (6.34 Tesla). Typical peaks of 31P-spectra of hamster liver showed a tendency for PDE/beta-ATP, alpha-ATP/beta-ATP and gamma-ATP/beta-ATP to decrease among the irradiated group compared with the control group. However, there were no significant differences in PME/Pi, beta-ATP/Pi or (alpha-ATP-beta-ATP)/beta-ATP between the control group and irradiated group. With regard to intracellular pH and PDE/beta-ATP, a decreasing tendency was noted in the irradiated groups (p less than 0.05). There was no difference in the signal intensity of T1WI and T2WI on 1H-MRI, between these two groups. Pathologically, the irradiated group showed minor hemorrhage and edema in the liver, and subcapsular hematoma. The results obtained from 31P-MRS clearly showed the metabolic changes and were correlated well with the histological findings, but MRI was not capable of providing close visualization of post-ESWL liver damage.(ABSTRACT TRUNCATED AT 250 WORDS)


[Transcatheter embolization for huge pulmonary arteriovenous fistula using metallic "spider" and spring embolus--application of hand-made metallic "spider" using partial monorail technique].

We performed transcatheter embolization in two cases with huge pulmonary arteriovenous fistula (AVF) using a metallic "spider" and spring embolus. Conventional spring embolus or detachable balloon could not be used in these cases. Metallic spider was indicated for pulmonary AVF with a feeding artery diameter of more than 16 mm to prevent embolus passing through the AVF. In the first case, we used large handmade metallic spiders of 25 mm in diameter followed by embolization by numerous spring coils. At that time, a partial monorail technique was newly devised to carry the large metallic spider into the feeding artery, otherwise the spider could not pass into a 9F catheter. After embolization, symptoms and PaO2 in arterial blood improved remarkably in both cases. In the second case, a spring coil migrated into the normal pulmonary artery, but no infarction resulted. In conclusion, the metallic spider was very useful for embolization of hugee pulmonary AVF to avoid the embolus passing through and to tangle spring coils together with it. If commercially available "spiders" are too small, ones can be made easily.


[MR imaging of corpus callosal injuries].

The MR imaging and CT findings of corpus callosal injury were analyzed in 32 of 224 patients with acute head injuries. MR imaging was more sensitive than CT in the detection of callosal injuries. All 9 hemorrhagic lesions were visualized on both MR imaging and CT. Fifteen of 23 nonhemorrhagic lesions were not visualized on CT, although all nonhemorrhagic lesions were visualized on MR imaging. Twenty-four lesions of the corpus callosum were located in the splenium, but no lesion was located in the rostrum. Diffuse axonal shear injuries were visualized in 25 patients with callosal injury as associated traumatic lesions. Twenty-three patients with callosal injury had low initial Glasgow Coma Scale scores (less than 9), but 9 patients had high scores. Associated diffuse axonal shear injuries, especially in the brain stem could be a possible explanation for this difference. MR imaging is useful to detect traumatic lesions of the corpus callosum.


[Experimental and clinical study of arterial damage induced by anti-cancer drug infusion].

In order to reduce the arterial damage following arterial chemo-infusion, arterial reaction to anti-cancer drugs and Corticosteroid were studied experimentally and clinically. In experiment, chemo-infusions (Mitomycin C, Adriamycin, Cisplatin) with or without Corticosteroid were carried out into the auricular and femoral arteries of rabbits, and the arterial changes were examined angiographically and histopathologically. The histologic examination showed the damages of various degrees characterized by intimal edema with pyknosis of endothelial cells, thrombus formation and detachment of intimal layer. The degree and frequency of the damage increased as the drug dose and concentration increased. However, higher blood flow and Corticosteroid could reduce the damages in some degrees. Clinically, bronchial arterial infusion of Cisplatin with or without Corticosteroid were studied. In conclusion, when angiography following ACI reveals narrowing and/or irregularity of the target artery, reduction of drug concentration and dose as well as elongation of infusion intervals are advised.


[MR imaging of the seminal vesicles--correlations between age, size and signal intensity].

MR findings of 82 cases without seminal vesicle lesions were retrospectively evaluated to assess the mutual correlations between age, size and signal intensity of the seminal vesicle. MR findings of 31 cases of carcinomatous invasion to the seminal vesicle were also evaluated as a control group for low signal intensity area-study. 1.5T superconductive unit (Toshiba 2000FX-II) was used to obtain T1-weighted (SE500/13) and T2-weighted (SE2500/80) images. Significant correlations were noted between age, short-axis diameter of the seminal vesicle and global signal intensity of the seminal vesicle on T2-weighted images. In the study of low signal intensity area on T2-weighted images, diffuse strand-like low signal intensity areas were occasionally seen in cases without seminal vesicle lesions. On the other hand, low signal intensity areas in cases of carcinomatous invasion to the seminal vesicle tended to be more localized and nodular. MR evaluation of cases without seminal vesicle lesions is important not only to delineate physiological changes of the seminal vesicle, but also to avoid erroneous MR-diagnosis of seminal vesicle lesions.


[Acute non-opacified dissection of the ascending thoracic aorta--significance of retrograde dissection and re-dissection].

Thirteen cases of acute aortic dissection with non-opacified false lumen of the ascending aorta were examined by CT and other imaging modalities. On the basis of the initial CT findings, these cases were classified into two types; one was pure non-opacified dissection not associated with opacified false lumen (Type N, n = 7), the other was non-opacified dissection of the ascending aorta associated with opacified false lumen of the following aorta (Type N+O, n = 6). On examining the relation between the entry site and the false lumen in Type N+O, the dissection of the ascending aorta was considered to be retrograde. Retrograde dissection seemed to be an important factor in the development of the non-opacified dissection of the ascending aorta. During the follow-up period, re-dissection in the ascending aorta occurred in four of the 13 cases (Type N = 3, Type N+O = 1). The re-dissection occurred within the first four weeks in all of them, and the diagnosis of re-dissection was possible at its early stage. In one case, ulcerlike projection (ULP) was detected by aortography. In another case, ULP was identified by cine-MR imaging. Contrast CT also revealed enlargement and small opacification of the false lumen. In two other cases, similar CT findings were observed. Three of the four patients recovered by surgical treatment. One died the day after the diagnosis of re-dissection. Early diagnosis and earliest possible surgical intervention for re-dissection were considered necessary to save the patients with re-dissected false lumen in the ascending aorta. Close observations with several imaging modalities, mainly CT examination, should be paid in the patients with non-opacified dissection of the ascending aorta for at least four weeks after the onset of dissection.


[Conformation radiotherapy of carcinoma of the prostate].

During the period from 1975 to 1989, 84 patients with carcinoma of the prostate were treated with conformation radiotherapy at Tokyo Metropolitan Komagome Hospital. The radiation field encompassed only the area of the prostate gland; it did not include the pelvic lymph nodes. The clinical stage of the 84 tumors was 22 in Stage A, 31 in Stage B, 15 in Stage C and 16 in Stage D. The average age of patients was 73.4 years, with range of 54 to 88 years. The average dose to the tumor was 65.7 Gy, with range of 60 Gy to 70 Gy. Hormone therapy was applied to 42 cases. The 5- and 10-year cumulative survival rates were 90.7% and 70.5% for Stage A, 41.7% and 26.7% for Stage B, 48.9% and 48.9% for Stage C, and 32.6% and 0% for Stage D, respectively. The 5-year cause-specific cumulative survival rates were 100% for Stage A, 92.3% for Stage B, 65.0% for Stage C and 40.3% for Stage D, respectively. Patients with poorly differentiated adenocarcinomas or undifferentiated carcinomas showed poorer survival than those with well-differentiated carcinoma. Only 7 cases suffered in-field recurrence, and 2 cases suffered recurrence at pelvic lymph nodes. Acute reactions were noted in 13 cases. Late complications following treatment were acceptable. Mild to moderate complications were recognized in 2 cases, but neither patient required surgery. In conclusion, our data suggest the advantage of the conformation technique applied to radiation therapy for carcinoma of the prostate.


Targeting of the creatine kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors.

Replacement vectors with genomic DNA originating from different mouse strains were used to introduce site-specific mutations into the creatine kinase M (CKM) gene of mouse embryonic stem (ES) cells. Here we demonstrate that in mouse strain 129-derived ES cells, the gene is at least 25-fold more efficiently targeted with an isogenic, 129-derived vector (129-pRV8.3) than with a nonisogenic, BALB/c-specific vector (BALB/c-pRV8.3). The two targeting constructs were identical except for allelic differences which were typed by partial sequencing. These included base pair mismatches (2%) and a polymorphic [GTC]-repeat length variation. Both in separate transfections as well as in cotransfections with mixed vectors, homologous disruption of the CKM gene resulted uniquely from the 129-isogenic DNA. Our data confirm earlier observations on requirements for homologous recombination in pro- and eukaryotic systems and indicate that targeting of the CKM locus is highly sensitive to small sequence differences between cognate segments in the endogenous and incoming DNA.


Thioredoxin regulates the DNA binding activity of NF-kappa B by reduction of a disulphide bond involving cysteine 62.

A role for redox regulation in activation of the NF-kappa B transcription factor was suggested by the observation that DNA binding activity of free protein, but not preformed DNA-protein complex, is inhibited by -SH modifying agents but enhanced by reducing agents. Mutagenesis of conserved cysteine residues in the p50 subunit identified amino acid 62 as being important for DNA binding, as a serine substitution at this position reduces DNA binding affinity, but renders the protein insensitive to -SH modifying agents. DNA binding activity of the wild type protein but not the amino acid 62 mutant was also stimulated by thioredoxin while detection of disulphide cross linked dimers in p50 but not the amino acid 62 mutant suggests that thioredoxin stimulates DNA binding by reduction of a disulphide bond involving cysteine 62. The physiological relevance of these findings was supported by the observation that cotransfection of a plasmid expressing human thioredoxin and an HIV LTR driven reporter construct resulted in an NF-kappa B dependent increase in expression of the reporter gene. Thus modification of p50 by thioredoxin, a gene induced by stimulation of T-lymphocytes in parallel with NF-kappa B translocation, is a likely step in the cascade of events leading to full NF-kappa B activation.


In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells.

We describe a process for the identification of mRNAs within single cells, as demonstrated with the immunoglobulin (Ig) variable region (V) genes of two mouse hybridoma cell lines and the bcr-abl fusion gene of the human K562 myeloid leukaemia line. The cells were fixed and permeabilised, the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction (PCR). After using fluorescent PCR primers, the amplified DNA could be detected within the cells as demonstrated by confocal fluorescence microscopy and flow cytometry. Furthermore the amplified Ig VH and VL DNA could be assembled within the same cell using suitable PCR primers. We detected no cross-contamination of amplified DNA between cells: the DNA isolated from mixtures of two hybridoma cell lines (B1-8 and NQ10/12.5) treated to in-cell PCR and assembly, was shown by cloning to correspond to the combinations of VH and VL genes of the parent hybridomas. We forsee diverse applications of in-cell assembly by PCR, especially for the analysis of the combinations of chains of rearranged Ig or T cell receptor (TCR) V-genes in a population of cells, and the construction of human antibodies from the V-genes of immune B-lymphocytes.


Effects of antioestrogens on the DNA binding activity of oestrogen receptors in vitro.

We have investigated the effect of a series of steroidal oestrogen antagonists, related to ICI 164,384, on the DNA binding activity of mouse oestrogen receptors expressed in insect cells. The analogues possess different side chains at the 7-position of the B ring in the steroid. Inhibition was observed when the length of the side-chain was 15-16 carbon atoms but not 10 or 20 carbon atoms and only when the 7 alpha isomer was used. The DNA binding activity of receptors expressed in COS-1 cells was also inhibited after extended periods of incubation with antioestrogens but not that of the human receptor in breast cancer cell-extracts. We have proposed that ICI 164,384 might disrupt receptor dimerisation, and therefore the variation in its ability to inhibit DNA binding activity may reflect differences in dimer stability. Since the DNA binding activity of in vitro translated receptors was inhibited when they were translated in the presence of the antioestrogen we suggest that ICI 164,384 might prevent the formation of receptor dimers without necessarily being able to disrupt preformed dimers.


Structure of the human DNA ligase I gene.

The gene encoding DNA ligase I, the major DNA ligase activity in proliferating mammalian cells, maps to human chromosome 19q13.2-13.3. We have determined the complete structure of the gene, which is composed of 28 exons spanning 53kb on this chromosome. The first exon is untranslated, and utilises a GC dinucleotide instead of the canonical GT splice donor. The 5' flanking region lacks a TATA box and is highly GC-rich, as is characteristic of a 'housekeeping' gene. In common with the promoters of genes encoding other DNA replication enzymes, such as DNA polymerase alpha, the 5' flanking region of the DNA ligase I gene contains recognition elements for several transcription factors which may mediate increased expression in quiescent cells in response to growth factors.


Role of an upstream open reading frame in the translation of polycistronic mRNAs in plant cells.

The influence of an upstream small open reading frame (URF) on the translation of two consecutive coding regions on an eukaryotic mRNA was studied. The cis effects of leader length, URF length, the sequences of the URF and neighboring regions, and the trans effects of the Cauliflower mosaic virus transactivator (TAV) were analyzed. Translation efficiency of the immediate downstream open reading frame (ORF) decreased with increasing URF length. Short URFs did not drastically inhibit translation of immediate downstream ORFs but supported far downstream translation in the presence of TAV. In the latter case, the optimal URF length was 30 codons.


The vacuum UV CD spectra of G.G.C triplexes.

Vacuum UV circular dichroism (CD) spectra were measured down to 175 nm for d(C)10, d(G)10, the d(G)10.d(C)10 duplex, and the d(G)10.d(G)10.d(C)10 triplex. A CD difference spectrum was calculated for d(G)10.d(C)10 giving the change in CD induced by forming the duplex from d(G)10 and d(C)10. The d(G)10.d(G)10.d(C)10 CD difference spectrum gave the CD induced by triplex formation from binding of d(G)10 to the d(G)10.d(C)10 duplex. In the near-UV, the d(G)10.d(C)10 and d(G)10.d(G)10.d(C)10 difference spectra resembled the difference spectrum for poly[r(G).r(C)] (Biopolymers 29, 325-333). This similarity may be an indication of similar purine base stacking. The d(G)10.d(G)10.d(C)10 vacuum UV difference spectrum had a negative band at 195 nm and a positive band at 180 nm, making it similar to difference spectra for homopolymer triplexes containing T.A.T and U.A.U triplets (Nucl. Acids Res. 19, 2275-2280). The appearance of these bands in difference spectra should be good indicators of triplex formation. The complementary oligonucleotides c-mycI d(CCCCACCCTCCC) and c-mycII d(GGGAGGGTGGGG) are part of the regulatory sequences of the human c-myc gene. G.G.C rich triplexes formed by binding c-mycII or c-mycIII d(GGGGTGGGTGGG) to the c-mycI.c-mycII duplex had CD difference spectra similar to that of d(G)10.d(G)10.d(C)10 in both the vacuum UV and near UV regions, indicating similar triplet structures.


Transcriptional induction of IFN-gamma-responsive genes is modulated by DNA surrounding the interferon stimulation response element.

The 9/27 and GBP mRNAs are both inducible by Interferon-gamma (IFN-gamma). The promoters of both genes contain an Interferon Stimulation Response Element (ISRE), but while the GBP gene is strongly induced transcriptionally by IFN-gamma the response of the 9/27 promoter is very weak. We investigated the molecular basis for this difference. The different IFN-gamma-responsiveness was found to have more than one reason. First, 9/27 promoter DNA was unable to bind the Gamma Interferon Activation Factor (GAF) with a single high affinity site. It efficiently competed for the association of the GAF with the GBP promoter but this competition was due to the presence of two low affinity sites, the ISRE and an ISRE-like sequence, suggesting that the GAS and ISRE, though both having clear preferences for specific proteins, may nevertheless share a certain degree of structural homology. Second, the 9/27 and GBP ISREs differed markedly in their affinities for regulatory proteins (ISGFs 1,2,3) and the GBP ISRE was more potent in mediating IFN-gamma-induced promoter activity in transient transfection. Third and most importantly, however, the strong difference between the IFN-gamma response of the two promoters was mainly due to the sequences surrounding the ISRE: the positive-acting GAS on one side and sequences with silencing properties 5' and 3' of the 9/27 ISRE on the other side. The data thus show mechanisms to both up- and down-regulate the activity of the ISRE.


A role for the human single-stranded DNA binding protein HSSB/RPA in an early stage of nucleotide excision repair.

The human single-stranded DNA binding protein (HSSB/RPA) is involved in several processes that maintain the integrity of the genome including DNA replication, homologous recombination, and nucleotide excision repair of damaged DNA. We report studies that analyze the role of HSSB in DNA repair. Specific protein-protein interactions appear to be involved in the repair function of HSSB, since it cannot be replaced by heterologous single-stranded DNA binding proteins. Anti-HSSB antibodies that inhibit the ability of HSSB to stimulate DNA polymerase alpha also inhibit repair synthesis mediated by human cell-free extracts. However, antibodies that neutralize DNA polymerase alpha do not inhibit repair synthesis. Repair is sensitive to aphidicolin, suggesting that DNA polymerase epsilon or delta participates in nucleotide excision repair by cell extracts. HSSB has a role other than generally stimulating synthesis by DNA polymerases, as it does not enhance the residual damage-dependent background synthesis displayed by repair-deficient extracts from xeroderma pigmentosum cells. Significantly, when damaged DNA is incised by the Escherichia coli UvrABC repair enzyme, human cell extracts can carry out repair synthesis even when HSSB has been neutralized with antibodies. This suggests that HSSB functions in an early stage of repair, rather than exclusively in repair synthesis. A model for the role of HSSB in repair is presented.


Identification of a novel downstream binding protein implicated in late-phase-specific activation of the adenovirus major late promotor.

The adenovirus major late promotor (MLP) is induced to very high levels after the onset of the viral DNA replication. Previous studies have identified sequence elements located downstream of the MLP startsite (DE1, between +85 and +98; DE2, between +100 and +120) implicated, together with the upstream promoter element, in this late-phase-specific transcriptional activation. One protein (DEF, now renamed DEF-A), induced during the late phase of viral infection, has been identified and shown to bind to the DE1 element (Jansen-Durr et al., 1989, J. Virol. 63, 5124-5132). Here we report about a distinct late-phase-specific protein (DEF-B) and its interactions with DEF-A. DNA-binding studies reveal that DEF-B interacts with the 5' part of DE2 (DE2b), whereas DEF-A, besides its interaction with DE1, also binds to the 3' portion of DE2 (DE2a), but with a lower affinity than for DE1. Furthermore, when added together, DEF-A and DEF-B cooperatively assemble onto the DE2 element as a heteromeric complex which is substantially more stable than the complexes formed by each protein alone. Using an in vivo transcriptional assay of the MLP, we show that DEF-A and DEF-B both have intrinsic transactivating properties.


Extraordinarily stable mini-hairpins: electrophoretical and thermal properties of the various sequence variants of d(GCGAAAGC) and their effect on DNA sequencing.

A small DNA fragment having a characteristic sequence d(GCGAAAGC) has been shown to form an extraordinarily stable mini-hairpin structure and to have an unusually rapid mobility in polyacrylamide gel electrophoresis, even when containing 7M urea. Here, we have studied the stability of the various sequence variants of d(GCGAAAGC) and the corresponding RNA fragments. Many such sequence variants form stable mini-hairpins in a similar manner to the d(GCGAAAGC) sequence. The RNA fragment, r(GCGAAAGC) also forms a mini-hairpin structure with less stability. The DNA mini-hairpins with GAAA or GAA loop are much more stable than DNA and RNA mini-hairpins with other loop sequence so far as has been examined. The stability difference between DNA and RNA mini-hairpins may be deduced to the stem structures formed by DNA (B form) and RNA (A form). The stable hairpins consisting of the GCGAAAGC sequence cause strong band compression on the sequencing gel. This phenomenon should be carefully considered in DNA sequencing.


Purification and characterization of DNA ligase I from the trypanosomatid Crithidia fasciculata.

A DNA ligase has been purified approximately 5000-fold, to near homogeneity, from the trypanosomatid Crithidia fasciculata. The purified enzyme contains polypeptides with molecular masses of 84 and 80 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both polypeptides formed enzyme-adenylate complexes in the absence of DNA, contained an epitope that is highly conserved between human and bovine DNA ligase I and yeast and vaccinia virus DNA ligases, and were identified in fresh lysates of C. fasciculata by antibodies raised against the purified protein. Hydrodynamic measurements indicate that the enzyme is an asymmetric protein of approximately 80 kDa. The purified DNA ligase can join oligo(dT) annealed to poly(dA), but not oligo(dT) annealed to poly(rA), and can ligate blunt-ended DNA fragments. The enzyme has a low Km for ATP of 0.3 microM. The DNA ligase absolutely requires ATP and Mg2+, and is inhibited by N-ethylmaleimide and by KCI. Substrate specificity, Km for ATP, and the conserved epitope all suggest that the purified enzyme is the trypanosome homologue of DNA ligase I.


Random mutations to evaluate the role of bases at two important single-stranded regions of genomic HDV ribozyme.

In elucidating function of two important single-stranded regions [SSrA (726-731 nt) and SSrB (762-766 nt)] derived mainly from three secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several random mutants at those two regions on the HDV88 molecule (683-770 nt) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among mutants, at the region SSrA, G726 was found to play an important role during cleavage reaction since substitutions of the base to A (mutant A20) or C (mutant A16) or U (mutant A23), reduced the ribozyme activity to very low levels suggesting the importance of G726 position. C763 at SSrB region was found to play a more significant role during catalysis than G726 (at region SSrA) since any substitutions at C763 completely inactivated the ribozyme. Other bases located in these two regions could be substituted to other bases at the expense of some self-cleavage activity. The results presented here together with our previous deletion analysis indicate that these two regions may play an important role during cleavage process.


Molecular cloning of RAD16, a gene involved in differential repair in Saccharomyces cerevisiae.

We have cloned the RAD16 gene of Saccharomyces cerevisiae and determined its nucleotide sequence. The gene complements the UV sensitivity of a rad16 mutant and restores the ability to repair the transcriptionally inactive HML alpha locus that is absent in this mutant. Disruption mutants that were constructed using the cloned gene are viable and UV sensitive and show no detectable growth defect. Moreover, such a mutant is deficient for repair of the HML alpha locus. The nucleotide sequence shows that the gene codes for a protein of 790 amino acids that has two potential zinc binding domains and shares homology with two other yeast proteins: the RAD54 gene product involved in recombinational repair and SNF2, a transcription factor that possibly functions in transcription activation through an interaction with chromatin components that allows access of other factors involved in transcription. The role of RAD16 in the repair of HML alpha might be to change the chromatin structure of silenced genes to provide access for excision repair enzymes.


Crosstalk between plasmid vegetative replication and conjugative transfer: repression of the trfA operon by trbA of broad host range plasmid RK2.

Previous deletion and complementation analysis has indicated that the region between trfA and kilBI (trbB) encodes trans-acting factor, designated trbA, required for conjugative transfer of broad host range plasmid RK2. In analysing the nucleotide sequence of this region we have discovered a gene encoding a 12 kDa polypeptide. The predicted amino acid sequence of this protein shows similarity at its C-terminal to KorA from the central control operon of RK2 and at its N-terminal to immunity repressor protein from phage phi 105 of Bacillus subtilis as well as the Sin protein of B. subtilis which regulates alternate developmental processes including sporulation, motility and competence. We show that TrbA represses transcription of both trfA (vegetative replication) and kilBI (trbB) (required for conjugative transfer and whose product has similarity to ComG, required for competence of B. subtilis) and may help to coordinate expression of both sets of functions. This region has similarities to some temperate bacteriophage immunity regions in modulating divergent transcription required for alternative means of propagation.


Enhancement of ribosomal frameshifting by oligonucleotides targeted to the HIV gag-pol region.

The pol gene of all retroviruses is expressed as a gag-pol fusion protein which is proteolytically processed to produce all viral enzymes. In the human immunodeficiency virus (HIV), the gag and pol genes overlap by 241 nucleotides with pol in the -1 phase with respect to gag. The gag-pol fusion is produced via a -1 ribosomal frameshifting event that brings the overlapping, out-of-phase gag and pol genes into translational phase. Frameshifting occurs at a so called 'shift site' 8-10 nucleotides upstream of a hairpin loop which may play a role in the regulation of frameshifting. We have fused this region of HIV-1 to the 5' end of the firefly luciferase reporter gene in order to quantitatively measure ribosomal frameshifting both in cells and by in vitro translation. A series of 2'-O-methyl oligonucleotides was designed to specifically bind the sequences which flank the gag-pol hairpin. Ribosomal frameshifting is enhanced up to 6 fold by those oligonucleotides which bind the area just 3 to the stem. Oligonucleotides which bind 5' to the stem have no effect on frameshift efficiency. In addition, we have constructed a series of fusion genes which mimic the effect of the bound oligonucleotides with intramolecular hairpins. The results suggest that increasing RNA secondary structure downstream of the shift site increases the frequency of ribosomal frameshifting, and that this effect can be mimicked by antisense oligonucleotides.


Evidence for a HeLa cell splicing activity that is necessary for activation of a regulated adenovirus 3' splice site.

Adenovirus late region 1 pre-mRNA splicing is temporally regulated during a lytic infection at the level of alternative 3' splice site usage to produce two mRNAs; the 52,55K and IIIa mRNAs which utilize the proximal and distal 3' splice sites, respectively. In vivo, the 52,55K mRNA is produced both early and late after infection, while IIIa is produced exclusively late in infection. Uninfected HeLa cell nuclear extracts, prepared with a low salt (0.4-0.5 M) or high salt (0.6 M and higher) wash, differed in their ability to splice 52,55K, IIIa and beta-globin transcripts. 52,55K and beta-globin precursors were spliced with similar efficiency in the low and high salt extract, while the IIIa mRNA was generated only in the high salt extract. Using the beta-globin pre-mRNA, no kinetic differences between the two types of extracts were observed. Nor were there any significant differences in the snRNA composition. The IIIa splicing activity did not appear to correlate with U2AF and pPTB levels. Our results suggest that a cellular trans-acting factor(s), which is required for adenovirus IIIa 3' splice site activation, is solubilized only at high salt concentrations.


Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts.

To study the possibility of gene rescue in plants by direct gene transfer we chose the Arabidopsis mutant GH50 as a source of donor DNA. GH50 is tolerant of chlorsulfuron, a herbicide of the sulfonylurea class. Tobacco protoplasts were cotransfected with genomic DNA and the plasmid pHP23 which confers kanamycin resistance. A high frequency of cointegration of the plasmid and the genomic DNA was expected, which would allow the tagging of the plant selectable trait with the plasmid DNA. After transfection by electroporation the protoplasts were cultivated on regeneration medium supplemented with either chlorsulfuron or kanamycin as a selective agent. Selection on kanamycin yielded resistant calluses at an absolute transformation frequency (ATF) of 0.8 x 10(-3). Selection on chlorsulfuron yielded resistant calluses at an ATF of 4.7 x 10(-6). When a selection on chlorsulfuron was subsequently applied to the kanamycin resistant calluses, 8% of them showed resistance to this herbicide. Southern analysis carried out on the herbicide resistant transformants detected the presence of the herbicide resistance gene of Arabidopsis into the genome of the transformed tobacco. Segregation analysis showed the presence of the resistance gene and the marker gene in the progeny of the five analysed transformants. 3 transformants showed evidence of genetic linkage between the two genes. In addition we show that using the same technique a kanamycin resistance gene from a transgenic tobacco could be transferred into sugar beet protoplasts at a frequency of 0.17% of the transformants.


Atomic force microscopy of reovirus dsRNA: a routine technique for length measurements.

Atomic force microscopy (AFM) was used to image reovirus double stranded RNA (dsRNA) deposited from diluted buffer solution onto a chemically treated mica surface. This procedure allows AFM images of dsRNA molecules to be obtained with a quality close to that obtained with conventional electron microscopy. The length of the molecules were measured directly on a computer display using the digitally acquired images. The lengths of the molecules varied between 0.2 and 1.8 microns. Statistical analysis showed a multimodal distribution with clear maxima at 0.4, 0.65 and 1.05 microns. These data are in a good agreement with those obtained by electron microscopy and gel electrophoresis.


Intronic sequence with both negative and positive effects on the regulation of alternative transcripts of the chicken beta tropomyosin transcripts.

The chicken beta tropomyosin gene generates three major transcripts by alternative splicing. A pair of internal exons are spliced in a mutually exclusive manner and their utilisation is developmentally regulated. Exon 6A and exon 6B are used respectively in myoblasts and myotubes during the process of differentiation of muscle cells. We have previously reported that, in myoblasts, exon 6B is skipped because of a negative regulation which involves intron as well as exon sequences. In this report, we describe a previously uncharacterized intronic element which is involved in the regulation of the splicing of both exons 6A and 6B. This cis-element is localized 37nt downstream of exon 6A and is approximately 30nt long. Its deletion, as well as modification of its sequence, results in the activation of the use of exon 6B and, at the same time, in the inhibition of the use of exon 6A. The mechanisms by which this region could act are further discussed.


Solid-phase synthesis of the nucleopeptide fragment H-Asp-Ser[pAAAGTAAGCC]-Glu-OH from the nucleoprotein of Bacillus subtilis phage phi 29.

The naturally occurring DNA-nucleopeptide H-Asp-Ser[5'-pAAAGTAAGCC-3']-Glu-OH was prepared via a solid-phase phosphite triester approach using N-2-(tert-butyldiphenylsilyloxymethyl)benzoyl protected nucleosides. The oligonucleotide was linked via the extremely base-labile oxalyl ester anchor to the solid support.


Intron 1 and the 5'-flanking region of the human thymidylate synthase gene as a regulatory determinant of growth-dependent expression.

We have determined the regulatory regions responsible for the growth-dependent expression of the human thymidylate synthase (TS) gene, using a set of minigenes constructed from segments of the human TS gene and the cDNA clone. Each construct was introduced stably into a TS-negative mutant of rat fibroblast 3Y1 cells. By serum-restricted synchronization of the cloned transformant cells, we found that a minigene with the genomic 5'-flanking region and intron 1 without other introns were sufficient for the normal extent and pattern of S-phase specific expression at the levels of both mRNA and enzymatic activity. In contrast, a TS cDNA clone driven by an SV40-based expression vector showed constitutive expression. Insertion of intron 1 into the cDNA clone in the normal location, or replacement of the viral 5'-promoter region of the cDNA clone by the genomic 5'-flanking sequence converted the constitutive expression to the S-phase dependent one, but only partly, that is, coexistence of the two regions were required for the normal expression. Results obtained by nuclear run-on assay suggested that posttranscriptional controls are also involved in this regulation in consistent with our previous results with the bona fide human TS gene.


Exon sequences distant from the splice junction are required for efficient self-splicing of the Tetrahymena IVS.

The presence of a natural rRNA secondary structure element immediately preceding the 5' splice site of the Tetrahymena IVS can inhibit self-splicing by competing with base pairing between the 5' exon and the guide sequence of the IVS (P1). Formation of this alternative hairpin is preferred in short precursor RNAs, and results in loss of G-addition to the 5' splice site. Pre-rRNAs which contain longer exons of ribosomal sequence, however, splice rapidly. As many as 146 nucleotides of the 5' exon and 86 nucleotides of the 3' exon are required for efficient self-splicing of Tetrahymena precursors. The presence of nucleotides distant from the 5' splice site apparently alters the equilibrium between the alternative hairpins, and promotes formation of active precursors. This effect is dependent on the specific sequences of the ribosomal pre-RNA, since point mutations within this region reduce the rate of splicing as much as 50-fold. This system provides an opportunity to study the way in which long-range interactions can influence splice site selection in a highly structured RNA.


Characterization of Arabidopsis thaliana telomeres isolated in yeast.

In an effort to learn more about the genomic organization of chromosomal termini in plants we employed a functional complementation strategy to isolate Arabidopsis thaliana telomeres in the yeast, Saccharomyces cerevisiae. Eight yeast episomes carrying A. thaliana telomeric sequences were obtained. The plant sequences carried on two episomes, YpAtT1 and YpAtT7, were characterized in detail. The telomeric origins of YpAtT1 and YpAtT7 insert DNAs were confirmed by demonstrating that corresponding genomic sequences are preferentially degraded during exonucleolytic digestion. The isolated telomeric restriction fragments contain G-rich repeat arrays characteristic of A. thaliana telomeres, as well as subterminal telomere-associated sequences (TASs). DNA sequence analysis revealed the presence of variant telomeric repeats at the centromere-proximal border of the terminal block of telomere repeats. The TAS flanking the telomeric G-rich repeat in YpAtT7 corresponds to a repetitive element present at other A. thaliana telomeres, while more proximal sequences are unique to one telomere. The YpAtT1 TAS is unique in the Landsberg strain of A. thaliana from which the clone originated; however, the Landsberg TAS cross-hybridizes weakly to a second telomere in the strain Columbia. Restriction analysis with cytosine methylation-sensitive endonucleases indicated that both TASs are highly methylated in the genome.


Trans replication and high copy numbers of wheat dwarf virus vectors in maize cells.

The replication of shuttle vectors derived from Wheat Dwarf Virus, a monopartite geminivirus, was studied in cultured maize endosperm cells, and in the Black Mexican Sweet (BMS) maize cell line. Using in vivo labeling and DNA methylation analysis, we showed that replication was initiated within 24 hrs after transfection, and did not require cell division in both cell lines. Copy numbers of 30,000 ds DNA molecules per cell were observed in endosperm cells after three days. The replication protein was shown to act in trans, since the wild type gene of the shuttle vector enabled replication-deficient vectors carrying mutated genes to replicate. These properties suggest that WDV may have similar applications in plants as SV40 in mammalian cells.


An ultraviolet light-induced crosslink in yeast tRNA(Phe).

The irradiation of native or unmodified yeast tRNA(Phe) by short wavelength UV light (260 nM) results in an intramolecular crosslink that has been mapped to occur between C48 in the variable loop and U59 in the T loop. Photo-reversibility of the crosslink and the absence of fluorescent photo adducts suggest that the crosslink product is a cytidine-uridine cyclobutane dimer. This is consistent with the relative geometries of C48 and U59 in the crystal structure of yeast tRNA(Phe). Evaluation of the crosslinking efficiency of the mutants of tRNA(Phe) indicates that the reaction depends on the correct tertiary structure of the RNA.


Hairpin and parallel quartet structures for telomeric sequences.

The role of thymine residues in the formation of G-quartet structures for telomeric sequences has been investigated using model oligonucleotides of the type d(G4TnG4), with n = 1-4. Sequences d(G4T3G4) and d(G4T4G4) adopt a G-quartet structure formed by hairpin dimerization in 70 mM NaCl as judged by a characteristic circular dichroism signature with a 295 nm positive and 265 nm negative bands while d(G4TG4) adopts a parallel G-quartet structure like d(G12) which exhibits a strong positive band at 260 nm and a negative band at 240 nm. The sequence d(G4T2G4) exhibits a mixture of both conformations. The stability of hairpin G-quartet structures decreases with decrease in the number of intervening thymine residues. Potassium permanganate, a single strand specific probe has been used to establish the presence of loops composed of T residues in the hairpin G quartet structures formed by the oligonucleotides d(G4TnG4) with n = 2-4 in 70 mM NaCl. The formation of hairpin G quartet structure for the above sequences is further supported by the enhanced electrophoretic mobility observed on non-denaturing polyacrylamide gels. Human telomeric sequence d(TTAGGG)4 which showed enhanced electrophoretic mobility like Tetrahymena telomeric sequence d(T2G4)4 also exhibited a characteristic CD spectrum for a folded-back G-quartet structure. A detailed model for G-quartet structure involving hairpin dimer with alternating syn-anti-syn-anti conformation for the guanine residues both along the chain as well as around the G tetrad with at least two thymine residues in the loop is proposed. Intermolecular association of short telomeric sequences reported here provides a possible model for chromosomal pairing.


Repression of the basal c-fos promoter by wild-type p53.

Mutations in the p53 gene are the most common genetic alterations observed in many inherited and sporadic forms of human cancer. Recent studies indicate that wild-type p53 may be involved in the regulation of gene expression. In the present report we examined the effect of p53 on the human c-fos promoter. Using a transient co-transfection assay we show that wild-type human p53, but not a transforming mutant of p53, negatively regulates the activity of the c-fos promoter in a dose-dependent manner. Promoter deletion analysis maps a sequence conferring p53 repression to the basal promoter region between nucleotides -53 and +42 relative to the cap site. In contrast, p53 strongly stimulates transcription when a sequence previously reported to bind p53 (TGCCT repeat) was inserted in front of the HSV-TK promoter driving CAT. These findings raise the question as to whether p53 may mediate its inhibitory effect on c-fos gene expression by interfering, directly or indirectly, with components of the basal transcriptional machinery.


Identification of regulatory elements within the minimal promoter region of the human endogenous ERV9 proviruses: accurate transcription initiation is controlled by an Inr-like element.

ERV9 is a low repeated family of human endogenous retroviral elements whose expression is mainly detectable in undifferentiated embryonal carcinoma NT2/D1 cells. In this report we have analyzed the minimal promoter region located within the ERV9 LTR. Using the transient CAT expression assay we have identified the minimal promoter region, which includes sequences spanning from -70 to +6 relative to the major transcription start site. Deletion analysis, primer extension mapping of the transcription start sites and DNA-protein interactions assays have allowed us to define two important regions within the ERV9 minimal promoter. One region located between -70 to -39 acts as a transcriptional activating sequence and contains an Sp 1 binding site. The second region from -7 to +6, which resembles an initiator element (Inr), was necessary for the correct transcription start site utilization, and binds to a regulatory protein. Cross-competition experiments using various Inr elements have indicated that the protein that binds to the ERV9 Inr element can be competed by the HIV-1 and TdT Inr sequences.


Zinc finger-DNA recognition: analysis of base specificity by site-directed mutagenesis.

Zinc fingers of the Cys2/His2 class are conserved 28-30 amino acid motifs that constitute an important and widespread family of eukaryotic DNA-binding domains. It is therefore of great interest to understand the rules that govern specific recognition of DNA by zinc fingers. The DNA-binding domain of the transcription factor Krox-20 consists of three zinc fingers, each of them making its primary contacts with a three-base pair subsite. We have performed a data base-guided site-directed mutagenesis analysis of Krox-20: nine derivatives were generated, in which one to three amino acid changes had been introduced within finger 2, at positions which were likely to affect the specificity of DNA recognition. The affinities of the different proteins for a panel of potential DNA binding sites were estimated by gel retardation assay. Six of the derivatives bound specific targets with affinities comparable to that of wild type Krox-20 for its consensus binding site. However, the specificity of recognition was dramatically modified at the expected bases, in a manner that could be explained by examining the newly introduced amino acids within the context of the overall finger/triplet interaction. These data provide new insights into the details of zinc finger-DNA interactions and, combined with the modular nature of zinc fingers, illustrate both the potential and the difficulties of utilising these motifs for designing DNA-binding proteins with novel specificities.


Multiple components are involved in the efficient joining of double stranded DNA breaks in human cell extracts.

We describe a rapid and efficient in vitro system for the rejoining of double stranded breaks in DNA based on extracts of human 293 cells. Using this system as an assay, we have separated the nuclear extract into several components involved in break rejoining. The unfractionated system can convert approx. 100% of the input DNA, linearized with a restriction enzyme, to high molecular weight material at low temperature (17 degrees C), and at the physiological temperature of 37 degrees C we have shown that competing activities in the extract can also act on the DNA template. We present the fractionation of the extract and the partial purification of a novel factor which will stimulate a crude rejoin activity and in addition increases the activity of purified DNA ligase I. We have also partially purified the break joining activity and show that the chromatographic properties do not directly correspond with the three DNA ligases previously described, indicating that the activity observed may not be due to a single enzyme species. By studying the rejoining of double stranded DNA breaks as a biochemical process, we have demonstrated that the efficient joining of such breaks requires factors in addition to DNA ligases.


Structure and expression of the nuclear gene coding for the plastid CS1 ribosomal protein from spinach.

The chloroplast ribosomal protein CS1 is an essential component of the plastids translational machinery involved in translation initiation. Southern analysis suggests that the corresponding nuclear gene is present in one copy in the spinach genome. We have isolated and sequenced the gene (rps1) to study its expression at the transcriptional level. The gene consists of 7 exons and 6 introns including an unusually large intron in the 5' coding region. No canonical TATA-box is found in the 5' upstream region of the gene. rps1 transcripts are detected early during germination and a significant accumulation is observed after the protrusion of the radicle. CS1 mRNAs are present in all organs of young seedlings although there are dramatic differences in the steady state level of the mRNAs between leaves and roots tissues. Transcripts accumulate independently of the presence or absence of light. Band shift analysis shows that the +1, -400 bp region of the gene can bind different sets of proteins isolated from roots and leaves nuclei. We suggest that the expression of the housekeeping plastid-related rps1 gene is regulated in a tissue-specific manner by transcriptional trans-acting factors.


Structure of the phenylalanyl-tRNA synthetase genes from Thermus thermophilus HB8 and their expression in Escherichia coli.

A 4459 bp long BamHI restriction fragment containing the two genes for the Thermus thermophilus HB8 phenylalanyl-tRNA synthetase was cloned in Escherichia coli and its nucleotide sequence was determined. The genes pheS and pheT encode the alpha- and beta-subunits with a molecular weight of 39 and 87 kD, respectively. Three conserved sequence motifs typical for class II tRNA synthetases occur in the alpha-subunit. Secondary structure predictions indicate that an arm composed of two anti-parallel alpha-helices similar to that reported for the E.coli seryl-tRNA synthetase may be present in its N-terminal portion. In the beta-subunit clusters of hydrophilic amino acids and a leucine zipper motif were identified, and several pronounced alpha-helical regions were predicted. The particular arginine and lysine residues in the N-terminal portion of the beta-subunit, which were found to participate in tRNA binding in the yeast and E.coli PheRSs, have their counterparts in the T.thermophilus protein. The 5'-portion of an open reading frame downstream of pheT was found and codes for a yet unidentified, extremely hydrophobic peptide. The pheST genes are presumably cotranscribed and translationally coupled. A novel type of a putative transcriptional terminator in Thermus species was identified immediately downstream of pheT and other Thermus genes. The genes pheS and pheST were expressed in E.coli.


Isolation and characterization of the chicken bcl-2 gene: expression in a variety of tissues including lymphoid and neuronal organs in adult and embryo.

The expression of human bcl-2 gene is de-regulated by t(14;18) translocation in most of follicular lymphoma. Recent studies indicated that the bcl-2 gene product has an ability to block apoptosis of hematopoietic cells. To facilitate the analysis of the role of this gene in normal development using an animal model, we have isolated and partially characterized the chicken homologue of human bcl-2 gene. The analysis of nucleotide sequence showed that the organization of the chicken bcl-2 gene is very similar to that of human bcl-2 gene. The primary transcript is spliced to encode a 25,687 dalton (233 a.a.) protein. The chicken Bcl-2 protein has two regions highly homologous to human Bcl-2 protein surrounding a totally non-homologous region. The expression of the chicken bcl-2 gene was analyzed in various chicken tissues. In the adult chicken, bcl-2 transcripts were detected in thymus, spleen, kidney, heart, ovary and brain, with the highest levels being detected in the thymus. However, the bursa of Fabricius, which is the site of early B cell development, expressed much less amounts of bcl-2 RNA. On the other hand, in embryo, the gene is extensively expressed in the bursa, as well as in muscle and the above tissues. Our findings indicate that a homologue of the human bcl-2 gene does exist in the chicken and that its expression is developmentally regulated in some tissues.


The promoter of the tgt/sec operon in Escherichia coli is preceded by an upstream activation sequence that contains a high affinity FIS binding site.

The tgt/sec operon in E. coli consists of five genes: queA, tgt, ORF12, secD, and secF. QueA and Tgt participate in the biosynthesis of the hypermodified t-RNA nucleoside Queuosine, whereas SecD and SecF are involved in protein secretion. Examination of the promoter region of the operon showed structural similarity to promoter regions of the rrn-operons. An upstream activation sequence (UAS) containing a potential binding site for the factor of inversion stimulation (FIS) was found. Gel retardation assays and DNaseI footprinting indicated, that FIS binds specifically and with high affinity to a site centred at position -58. Binding of FIS caused bending of the DNA, as deduced from circular permutation analysis. Various 5' deletion mutants of the promoter region were constructed and fused to a lacZ reporter gene to determine the influence of the UAS element on the promoter strength. An approximately two-fold activation of the promoter by the UAS element was observed.


Cationic lipid-mediated transfection of liver cells in primary culture.

We describe transfection of DNA into parenchymal and individual non-parenchymal cell populations from adult rat liver in early primary culture, using cationic lipid as the carrier. All cell populations were transfectable, although lipid requirements varied by cell type and, for hepatocytes, with the age of the culture. For hepatocytes in early primary culture (2-10 hours after plating), pure DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) was strikingly more effective than commercial formulations (Lipofectin or TransfectACE) containing components in addition to, or other than DOTMA. For hepatocytes fully adapted to culture (approximately 48 hours after plating), pure DOTMA and Lipofectin were similarly effective. Under optimal conditions, about 10% of hepatocytes expressed the transfected reporter gene. CAT expression in hepatocytes doubled from 48 hours to 7 days after transfection. The effect of culture substratum on transfection efficiency also was examined. The presence of basement membrane-like matrix (EHS gel) reduced uptake of the DNA-lipid complex. However, cells in early culture that were transfected on collagen and then replated on EHS gel, displayed significantly greater reporter gene activity than did cells maintained throughout on collagen. In contrast to hepatocytes, non-parenchymal cells (lipocytes, Kupffer cells and endothelial cells, respectively) were transfected most efficiently by Lipofectin; DOTMA alone was inactive. The methods described will facilitate studies of gene regulation in individual liver cell populations.


Blockage of polymerase-catalyzed DNA chain elongation by chemically modified cytosine residues in templates and the release of blockage for readthrough.

The Klenow fragment-mediated in vitro DNA elongation was inhibited by the presence of a class of modified cytosines in the template DNA, i.e., the N4-amino(and -methoxy)-5,6-dihydrocytosine-6-sulfonate residues. We have studied the mechanism of the blockage, using as templates bisulfite-hydrazine (and -methoxyamine)- modified single strand phage-M13mp2 DNA and synthetic oligonucleotides. Both N4-amino-5,6-dihydrocytosine-6-sulfonate and N4-methoxy-5,6-dihydrocytosine-6-sulfonate residues blocked the elongation at one nucleotide before these sites. In this blockage, the idling of polymerase at the lesion site due to its 3'-5' exonuclease action appears not to play a major role, because Sequenase that lacks the 3'-5' exonuclease activity still could not readthrough these sites. It seems possible that conformational distortion of the template near these sites is responsible for the blockage, because on conversion of this 5,6-dihydropyrimidine-6-sulfonate structure into a planar pyrimidine, a complete restoration of polymerase-readthrough resulted. In the presence of RecA and SSB proteins, the Klenow fragment was able to partially readthrough these sites. Since there was no decrease in the 3'-5' exonuclease activity during this readthrough, it seems that the binding of these proteins relaxes the distortion in the modified template to allow the polymerase to readthrough the lesion site. These sites on phage DNA can be lethal but also are capable of inducing C-to-T transitions. This observation suggests that these sites can be read by E. coli DNA polymerases in vivo with accompanying errors.