Immune changes in HIV-1 infection: significant correlations and differences in serum markers and lymphoid phenotypic antigens. Human immunodeficiency virus type 1(HIV-1) induces extensive immune cell alterations which can be detected by changes both in serum levels of soluble immune activation products and in several lymphoid phenotypic markers. The current studies were conducted in 70 HIV-1 seropositive subjects to determine whether changes among four important serum immune activation markers (neopterin, beta-2 microglobulin, soluble CD8, and soluble IL-2 receptor) and seven lymphoid phenotypic markers (CD38, HLA-DR, CD57, CD11b, CD45RA, leu8, and CD71) reflect similar or disparate aspects of immune pathology. On the basis of correlation coefficient calculation, four groups of related markers (Fig. 1) were identified: Group A, sIL-2R was related to group B where serum neopterin, beta 2M, sCD8 levels, and lymphocyte CD38 antigen expression correlated closely. Loss of CD45RA or Leu 8 antigens in group C correlated with group B and D markers increase. HLA-D in group D was a more distantly related immune activation marker. Phenotypic markers CD57, CD11b, and CD71 did not correlate with the immune activation processes reflected by the serum and phenotypic marker groups A-D. Correlations between serum and certain lymphoid phenotypic markers were generally stronger later in HIV-1 infection when CD4 levels were less than 500/mm3. This study provides information for selecting markers for investigating immune changes in HIV-1 infection and immune-related diseases. Many serum and lymphoid phenotypic markers reflect related aspects of immune dysregulation. However, some markers can indicate different aspects of disease.


[Analysis of prognosis-associated factors in pancreatic head and peri-ampullary cancer]. Between 1982-1990 we treated 461 patients with adenocarcinoma of the pancreas or the periampullary region. 125 (68 ductal pancreatic and 57 periampullary carcinomas) of these patients (27.1%) underwent resection. Hospital lethality was 3.6% (n = 4). Distribution of pT stages (UICC 1987) and frequency of complete resection (R0 vs. R1/R2) were significantly different between the periampullary and pancreatic tumors. This appeared to be due to the high frequency of pT1/2 periampullary tumors (49.2%) as compared to the prognostically equivalent pT1 tumors of the pancreas (1.9%). The absence of lymph node metastases significantly improved survival of periampullary tumors. This was not observed in tumors of the head of the pancreas. This data indicate that the poor prognosis of ductal pancreatic compared to periampullary cancer is primarily caused by their advanced stage at the time of diagnosis. In addition current resection techniques only inadequately respect the complex lymphatic drainage of the head of the pancreas.


[Risks and follow-up of choledocho-jejunostomy for nonresectable cancers of the head of the pancreas. A prospective study]. In 44 patients with palliative operative procedures due to non-resectable pancreatic cancer the preoperative risk factors and the intra- and postoperative course as well as the long-term results were prospectively investigated. Postoperatively 2 patients developed severe complications and 3 patients died. The median survival time was 5 months. The results of this investigation show, that the biliodigestive anastomosis offers a good palliation in patients with non-resectable pancreatic cancer. Indication for non-operative palliative procedure are patients in poor general condition with a life expectance of less than 4 months.


[Cryotherapy in rectal cancer. A palliative local tumor treatment]. Cryotherapy was applied to 182 rectal cancer patients in the Department of General Surgery, University of Ulm, between 4/1982 and 4/1991. Recipients of this tumor freezing therapy were patients whose general condition was bad, patients with an advanced inoperable carcinoma, patients with tumor recurrence and patients refusing operation. Rectal carcinomas, mostly in an advanced stage, were usually freezed several times. Only 4 patients with general inoperability reached the survival time margin of 5 years. In 18 patients local tumor destruction was possible by application of cryotherapy. In 80% of cases disagreeable tumor symptoms like tenesma, mucous discharge and oozing hemorrhages could be reduced or completely removed. Perianal pain and intense tumor bleeding could be relieved temporarily or definitely in only 50% of patients. An artificial anus could be avoided in 80% of cases by local tumor destruction/reduction or arrest of tumor growth. The mean survival time of patients with tumor recidivation was 11 months after onset of the recurrence. Tumor progression, incontinence and iatrogenic rectal perforation made an artificial anus necessary in 14 patients.


Supportive role of image analysis and DNA ploidy pattern in the diagnosis of thyroid tumors. To evaluate the supportive role of image cytometry and DNA ploidy pattern in the diagnosis of thyroid tumors, a preliminary study was performed on fine needle aspirates of 30 cases. Of these, 10 cases each were of colloid goiter, follicular neoplasm, and papillary carcinoma. The nuclear area and DNA value of 50 cells in each case were measured. The mean nuclear area in colloid goiter (69.50 + 12.62 sq.microns) was significantly lower than the mean nuclear area in a follicular neoplasm (88.71 + 15.51 sq.microns) (P less than 0.05). Similar differences between the mean nuclear area in colloid goiter and papillary carcinoma (124.0 + 12.27 sq microns) was also highly significant (P less than 0.001). The results obtained by image cytometry were compared with estimated DNA ploidy pattern of follicular cells from the same cases. All colloid goiter had mean nuclear area below 100 sq.microns with diploid DNA value. However, papillary carcinoma showed aneuploid DNA pattern in eight cases (80.0%), but mean nuclear area was above 100 sq.microns. A diagnostically useful finding obtained in two of the 10 cases of follicular neoplasm was the association of aneuploid DNA pattern with mean nuclear area of the follicular cells above 100 sq.microns indicating a high probability of carcinoma and thus demanding an urgent open biopsy. These cases were readily distinguished from other cases of the same category showing diploid DNA pattern and mean nuclear area of follicular cells below 100 sq.microns.


Identification of Acanthamoeba in bronchoalveolar lavage specimens. Acanthamoeba species infect humans occasionally and act as opportunistic pathogens in immunocompromised individuals. This study demonstrates the application of cytocentrifugation as an aid to identification of Acanthamoebae. In addition, certain staining procedures clearly optimized visualization of characteristic amoebic features. This was demonstrated by adding amoebae from laboratory cultures to bronchoalveolar lavage specimens. In preparations stained by the Papanicolaou, trichrome, or hematoxylin-eosin (H&E) procedures, the discrete deeper staining nucleolus was the most distinctive feature. The vacuolated cytoplasm also aided in the identification of amoebae. These features were less apparent and often distorted following staining of Acanthamoeba species with the Hema III and Giemsa procedures.


Assessment of a computer program to detect epileptiform spikes. This study compares an automated spike detection program to a group of 6 electroencephalographers. Since group members varied in experience, an expertise factor was devised to weight their scoring. EEGers underscored epileptiform events on 6 records in a manner analogous to the computer's storage of EEG segments. A summation of expertise factors was determined for every event. This sum was normalized and interpreted as a probability the event would be called a spike by a given EEGer. The performance of each scorer and of the computer at different amplitude thresholds was analyzed based on this probability. Higher rated scorers identified more subtle events. Lowering the threshold of the computer program produced a comparable increase in sensitivity. The increase in total events detected by the computer was linear over the range studied. While the proportion of false positive detections increased with lowering threshold, our readers have not found a moderate number of these distracting. We conclude that the computer system, while not as specific as an EEGer, can be as sensitive and can be a reliable screening editor for large amounts of monitoring data. On balance it is more effective than an EEGer for this limited purpose.


State dependent spike detection: validation. We present a formal validation of the method of state dependent spike detection; its principle and initial results were given in Gotman and Wang (1991). The method utilizes different procedures for elimination of non-epileptic transients according to state (active or quiet wakefulness, sleep stages); it also varies detection sensitivity according to state. Twenty new 100 min recordings were obtained from 20 unselected patients, covering active wakefulness, quiet wakefulness and various sleep stages. Results indicated that, while the total number of detections was reduced by 15%, true detections increased by 39% and false detections decreased by 60%. This significant improvement is due to the fact that wave morphology is now considered within its spatial context and particularly within a wide temporal context.


EEG changes in patients during the introduction of carbamazepine. This study evaluates the EEG changes during the standardized introduction of carbamazepine in 16 previously untreated neurological patients and their relationship to serum levels of carbamazepine and carbamazepine-10,11-epoxide. Therapy was started with a dosage of 400 mg carbamazepine b.i.d. and remained unchanged during the whole study period of 35 days. Frequency analysis of serial EEG records was performed by Fast Fourier Transformation. In comparison to the pretreatment period (1) the mean values of the total power and relative powers of the theta and delta bands increased and (2) the mean values of the relative power of the alpha band and the center frequency decreased. These changes were already established 3 days after the beginning of the treatment and remained constant during the observation period. There were marked interindividual differences. (3) There was no statistically significant correlation between serum levels of carbamazepine or carbamazepine-10,11-epoxide and the EEG parameters. Our results demonstrate that the degree of EEG change primarily reflects individual susceptibility to carbamazepine and its metabolite during the early stage of carbamazepine exposure and is not dose related.


Quantitative analysis of the EEG during tonic REM sleep--methodology. Frequency and amplitude characteristics of the clinical (waking) electroencephalogram (EEG) can be diagnostically useful in neuronal degenerative disorders such as Alzheimer's or other cortical dementias. However, interpretation of the clinical EEG may be limited by many factors, including movement and muscle artifacts and uncontrolled variations along the alert/drowsy continuum. Moreover, the clinical EEG involves subjective judgement by experts whose opinions often differ. In an effort to address these problems, we have developed a computer-automated technology that examines the digitized, all-night sleep EEG for frequency and amplitude characteristics of potential diagnostic relevance to Alzheimer's dementia. Robust time series analysis techniques and a modified power spectral analysis (Z-spectra) are used to suppress artifactual information and to automatically select samples of tonic REM sleep EEG. The spectra (amplitude vs. frequency relationship) of this specific EEG state is then assessed for diagnostically relevant information.


EEG markers of early Alzheimer's disease in computer selected tonic REM sleep. All night sleep/wake EEGs were examined for diagnostic features sensitive to early Alzheimer's disease (AD) using computer automated techniques. Thirty-nine mild AD patients and 43 normal controls underwent 9 h of EEG recording in the sleep laboratory. All-night EEGs were screened for ideal, low artifact tonic REM sleep using autoregressive and power spectral techniques. The frequency spectra during tonic REM sleep revealed a significant shift towards slower wave forms in AD vs. control subjects. Beta (greater than 12 Hz) was reduced and theta and delta (2-8 Hz) increased in AD compared to control groups. This frequency shift was demonstrated by several analytic techniques, including binned spectral energies and unique zones in the frequency spectra. Discriminant analyses using optimal binned EEG variables correctly classified 74% of AD and 98% of control subjects, and unique zone scores correctly classified 92% of AD and 95% of control subjects, indicating that these sleep EEG changes are apparently predictive of AD status.


Background EEG reactivity in auditory event-related potentials. Mental activity has influences on auditory event-related potentials (AERP) as well as on some EEG rhythms, notably the alpha rhythm. In this study, background reactivity (BR) of the EEG was investigated in the context of AERPs. Single responses of 14 healthy subjects were analysed in 3 reaction time AERP experiments of increasing difficulty: the first involved one type of tone delivered at random intervals, the second offered two types of tone (an "oddball" design), and the third offered 3 types of tone. Averages were formed and subtracted from the separate responses to reduce their contribution to the EEG. The 1526 msec EEG epoch was divided in 9 overlapping periods, and averaged power spectra were calculated for these periods. Changes in area of the delta, theta, alpha and beta bands in the course of the epoch were calculated. Background alpha and beta activity decreased following infrequent but not following frequent tones. The decrease was larger for more difficult tasks and reached a maximum in the period in which the AERP P3 and N3 peaks fell. Delta and theta powers showed increases rather than decreases; these could be attributed to the contribution of AERP activity to total EEG power. No clear relationships were found between the amount of background reactivity and peak latencies or reaction times. The occurrence of background reactivity, not apparent in an average, shows that the averaged AERP reveals only part of the EEG changes related to mental activity. Background reactivity proved to be more sensitive to task difficulty than P300 latency. The results are discussed in the context of the additive model of evoked potentials.


Delayed P3 event-related potentials (ERPs) in thalamic hemorrhage. Delayed P3 ERPs were recorded with an acoustic oddball paradigm in 5 patients affected by unilateral thalamic hemorrhage. P3 latencies in these 5 patients were above the normal mean latency of age-matched controls +2/+3 S.D.s. The P3 delay was persistent in serial follow-ups at 6 months from stroke. Normal P3 latencies were instead recorded in 4 patients with paramedian hemorrhage not involving or only partially involving the thalamus.


Event-related synchronization (ERS): an electrophysiological correlate of cortical areas at rest. Oscillations in the alpha and beta bands can display either an event-related blocking response or an event-related amplitude enhancement. The former is named event-related desynchronization (ERD) and the latter event-related synchronization (ERS). Examples of ERS are localized alpha enhancements in the awake state as well as sigma spindles in sleep and alpha or beta bursts in the comatose state. It was found that alpha band activity can be enhanced over the visual region during a motor task, or during a visual task over the sensorimotor region. This means ERD and ERS can be observed at nearly the same time; both form a spatiotemporal pattern, in which the localization of ERD characterizes cortical areas involved in task-relevant processing, and ERS marks cortical areas at rest or in an idling state.


Correlations between verbal memory performance and electrocorticographically determined suppression of electrical brain activity in intracarotid amobarbital tests. As a part of presurgical evaluation, bilateral intracarotid amobarbital procedures (IAPs) were performed in 42 patients (84 tests) with long-standing, medically intractable complex-partial seizures. During the IAPs, electrocorticographic (ECoG) recording was carried out via bilaterally implanted subdural electrodes. Five distinct patterns of suppression of electrical brain activity were observed: (1) an isoelectric line; (2) a burst-suppression pattern; (3) polyphasic waves; (4) high-voltage beta; and (5) low-voltage beta activity. Further, two types of specific reactions of the epileptic focus were detectable: (1) spike-burst-suppression patterns (SBS; 11 left; 7 right; 6 in both IAPs) and amobarbital-induced spikes (7 left; 4 right). ECoG suppression patterns as well as SBS and spike induction showed great variability in duration and overall occurrence. To determine the influence of these amobarbital-induced ECoG changes on results of IAP memory testing, performance in a verbal learning task was analyzed according to the ECoG patterns predominant during encoding. In left IAPs, it was found that SBS at the beginning of or during encoding had a significant negative effect on verbal memory. In right IAPs, verbal memory performance improved significantly with the decline of ECoG suppression. Hence, verbal memory performance in IAPs is significantly affected by specific ECoG suppression patterns and activation of the epileptic focus.


Rapid stream stimulation and the recognition potential. The "recognition potential" is an electrical response of the brain that occurs for recognizable, but not for non-recognizable, images. When a recognizable image evokes it, the more rostral of a pair of vertically oriented occipital electrodes reaches an initial positive peak at about 200-250 msec. Several properties distinguish this component of the evoked response from event-related potentials such as N2 or P3. A new method of stimulation was devised that evoked the recognition potential for recognizable images, but virtually no response of any kind for non-recognizable images. This was accomplished by presenting images at high rates. Chinese ideographs evoked it for subjects whose native language was Chinese, but not for subjects unfamiliar with that language. This showed that the recognition potential was not caused by differences in the physical attributes of the images per se. Instead, recognizability, as defined by a subject's individual learning experience, was the important factor.


Electrophysiological findings in a case of severe intrathecal baclofen overdose. Multimodality evoked potentials were examined in a case of serious accidental intrathecal baclofen overdose in a patient who suffered from severe spasticity due to a traumatic brain lesion. Electrophysiological findings during, before and after the intoxication were compared. Transcranial electrical stimulation up to 750 V did not evoke any responses in thenar muscles on the first day of intoxication. An improvement to normal values was observed within 3 days, paralleled by an amelioration of the patient's clinical condition. Cervical electrical stimulation was largely unaffected by baclofen. Median nerve somatosensory and brain-stem acoustic evoked potentials revealed few or no differences during intoxication compared to pre- and post-intoxication responses.


Demonstration of mismatch negativity in the monkey. In humans, deviant auditory stimuli elicit an event-related potential (ERP) component, termed "mismatch negativity" (MMN), that reflects the operation of a cortical detector of infrequent stimulus change. Epidural auditory ERPs were recorded from 3 cynomolgous monkeys in response to soft and loud clicks. "Oddball" loud or soft stimuli elicited a long-duration frontocentral negativity, peaking at approximately 85 msec, that was superimposed upon cortically generated obligatory ERP components. These data suggest that monkeys might serve as a heuristically valuable system in which to study the neurochemical and neuroanatomical substrates of early context-dependent ERP generation.


Turn and phase counts of individual motor unit potentials: correlation and reliability. Different turn algorithms are used for quantitative motor unit potential (MUP) analysis. To compare their retest reliability, 420 myopathic and neuropathic MUPs were recorded twice and the turn count of the first registration was correlated with that of the second. Reliability was best for the algorithm according to Willison as compared to the conventionally used algorithms based on amplitude criteria for 2 or 3 successive relative extrema. As demonstrated by discriminant analysis, an amplitude limit of 25 microV yielded more useful turn counts than a limit of 50 microV if myopathic MUPs had to be discriminated from normal MUPs. For this discrimination the turn count was superior to the phase count which did not further improve the discriminant model. This was different for the discrimination between normal and neuropathic MUPs. In this case, both parameters measured partly independent features of the MUP and had to be considered together.


The influence of muscle length on muscle fibre conduction velocity and development of muscle fatigue. The influence of muscle (vastus lateralis) length on the muscle fibre conduction velocity (MFCV) and on muscle fatigue was studied in 8 healthy volunteers. In experiment 1, the electromyographic (EMG) responses were evoked by electrical stimulation of the motor point and recorded by a surface electrode array aligned along the muscle fibre direction. The MFCV (determined by cross-correlation) was measured at knee flexions of 5 degrees (full extension), 45 degrees, 90 degrees and 120 degrees with 3 different extension torques. The MFCV declined with increasing muscle length and increased with increasing background torque at knee flexions from 5 degrees to 90 degrees. From 90 degrees to 120 degrees knee flexion of MFCV tended to increase. In experiment 2, the EMG activity at a static fatiguing contraction (80% MVC) was measured at 45 degrees and 90 degrees knee flexion. The EMG was measured until the subject gave up contracting the muscle (endurance). The largest increase in the RMS amplitude and the fastest decreases in the mean power frequency (MPF) and MFCV were found at 90 degrees flexion. The MVC at 45 degrees knee flexion was 35% lower than at 90 degrees and the time until endurance was approximately twice as long for the 45 degrees contraction. The results indicate that muscle length is an important parameter for the propagation velocity of action potentials and for the development of static muscle fatigue.


Nerve conduction in the hands of vibration exposed workers. Symptoms of peripheral neuropathy in the hands are common among workers using vibrating tools. The mechanism for this and its relation to carpal tunnel syndrome (CTS) was studied in workers exposed to vibration at their workplace (17), along with a control group of healthy construction workers with heavy manual work but without vibration exposure (10). Patients with uni- or bilateral CTS (11) and a group of healthy volunteers without manual work (9) were included for comparison. Median nerve conduction velocities were measured both over the carpal tunnel and in a more distal segment. Vibration exposed workers had similar conduction velocities to unexposed construction workers. The subgroup of vibration exposed patients with symptoms from the hands had normal conduction in the ulnar nerve but demonstrated a decrease in median nerve conduction comparable (but less pronounced) with the CTS group. On a group basis these results indicated that the median nerve is most vulnerable for hand-arm vibrations. However, the conduction defects were not pronounced enough to diagnose CTS in most individual cases.


Contralateral influences on triceps surae motoneuron excitability. In an effort to more fully investigate spinal reflex pathways in humans, we measured the isometric force-time curve of the tibial nerve H-reflex in 12 college age subjects. We also conditioned the reflex with a contralateral H-reflex stimulus or a contralateral tendon-tap, to ascertain the effects of crossed spinal segmental inputs on alpha motoneuron excitability. The conditioning stimulus preceded the test reflex by 10, 25, 40, 55, 70, 85, 100, 115, 130 or 145 msec. The results demonstrate that a conditioning tibial nerve H-reflex produced marked facilitation onto the contralateral triceps surae motoneurons, predominantly at longer-latency intervals. Conversely, a conditioning Achilles tendon-tap produced long-latency inhibition to the triceps surae. These results demonstrate that differential motoneuron excitability changes can be produced by electrical and mechanical conditioning stimuli. Moreover, these excitability changes may be long lasting and only appear after a relatively long latency. Several neurophysiological mechanisms are proposed to contribute to these changes.


Activation of the neck muscles from the ipsi- or contralateral hemisphere during voluntary head movements in humans. A reaction-time study. Reaction times (RTs) of EMG onset of agonist right and left sternocleidomastoid (SCM) and splenius (SPL) muscles in response to acoustic (AC) or unilateral somatosensory (SS) stimulation were measured in normal subjects, during head rotation (to the right or to the left) and flexion. No significant difference was present in the AC-RT of SCM muscles of the two sides between rotations or flexion. The same was true for the SPL. Under the tactile condition, in which the stimulus was delivered to one index finger, the RTs of both agonist muscles were shorter during head rotation toward the stimulus than away from it: the SCM contralateral to the stimulated finger was faster than the ipsilateral SCM, while the reverse was true for the SPL. During flexion, the SS-RTs of the SCM of both sides were similar, and similar in turn to the SCM-RT during rotation away from the stimulus. When the stimulus was delivered to the shoulder, the RT difference between the agonist SCMs disappeared. The delay in the activation of the SCM ipsilateral to the stimulated finger is compatible with the interhemispheric transmission time in the absence of callosal connection between the hand areas of the primary somatosensory cortex. The data favour the hypothesis that SCM activation during voluntary head rotation is controlled by the ipsilateral hemisphere.


Antagonist muscle inhibition before rapid voluntary movements of the human wrist. When a fast voluntary movement is performed from a background condition of sustained antagonist muscle activation, there is often a decrease in antagonist muscle activity before the onset of the first agonist muscle burst (AG1) that continues until the onset of the antagonist muscle burst (ANT). We studied how controlling the peak velocity, movement size, and the magnitude of antagonist muscle loading affected antagonist muscle inhibition (AntI). AntI was more pronounced during movements with lower velocity and greater size, and when performed in the direction of heavier background loads, but its variation could not be related to any single kinematic or kinetic variable in all circumstances. When AntI was larger, ANT was smaller, suggesting that AntI does not play a role similar to the premotor silence of the agonist seen before AG1 when the movement is made from a background of sustained agonist contraction. When AntI was larger, the size of AG1 was also smaller, showing that, according to the motor task, different levels of reciprocal inhibition and coactivation occur at the onset of the movement. Both AG1 and AntI produce force in the direction of the desired movement, and the central nervous system selects an appropriate balance between the two, using AntI when possible.


Motor responses evoked by magnetic brain stimulation in Huntington's disease. In 34 patients with manifest Huntington's disease (HD), and in 21 first-degree offspring without clinical signs or symptoms, the sizes, central motor latencies (CMLs) and variation in latencies of EMG responses (MEPs) following transcranial magnetic brain stimulation were studied in muscles of the upper and lower extremities. In subgroups of patients and their offspring median and tibial nerve somatosensory evoked potentials (SEPs) and electrically elicited long-loop reflexes (LLRs) in hand muscles were also investigated. Increased MEP thresholds were observed in 10% of the HD offspring, while CML, latency variability and MEP amplitudes always lay within normal range. In contrast, SEPs were abnormal in 33%. In HD patients MEPs were found to be abnormal in up to 72% of patients when all available response parameters were taken into consideration. MEP abnormalities correlated with the duration of motor symptoms and the severity of choreic motor activity. When both MEPs and SEPs were evaluated, abnormalities could be detected in 91% of all HD patients. We suggest that abnormal MEPs might reflect an altered excitability of the cortico-spinal system as a consequence of basal ganglia dysfunction, rather than a structural damage of the investigated descending pathways. To localize the pathological mechanism responsible for altered LLRs, a "loop analysis" was performed by recording LLRs, MEPs and SEPs in the same patients. Alterations of LLRs correlated best with abnormal SEPs and might therefore be explained by reduced somatosensory input to the motor cortex.


Electrical stimulation of motor cortex in experimental cortical ischaemia: pyramidal responses at C5 and the surface EMG. In 7 baboons maintained under propofol anaesthesia, pyramidal tract responses were related to the corresponding peripheral EMG evoked by electrical stimulation of the motor cortex under conditions of focal cortical ischaemia. Pyramidal responses were recorded epidurally at the C5 level and the EMG was recorded from the contralateral hand or foot muscle using subdermal needle electrodes. Cortical ischaemia was produced by transorbital occlusion of the common anterior cerebral artery, and regional cortical blood flow was measured by the hydrogen clearance method. In the normally perfused brain, the later I waves of the C5 response required a lower stimulus strength to elicit them than the earlier I1 wave. It was more difficult to record the EMG from the hand than from the foot following stimulation of the corresponding cortex even though the C5 responses were always obtained in both cases. With moderate ischaemia, the later I waves were selectively abolished, leaving the D and I1 waves. EMG amplitude was significantly correlated with cortical blood flow (r = 0.88, P less than 0.005), and the threshold of cortical flow for the EMG was 10-13 ml/100 g/min. Our results indicate that changes in amplitude of the late I waves and particularly of the EMG are sensitive indicators of cortical ischaemia.


Magnetic brain stimulation and brain size: relevance to animal studies. Magnetic brain stimulation (MBS) is widely used for the investigation of brain function in man, but there have been only a few reports of its safety in animals. These results were predominantly benign, but the effectiveness of stimulation in animals is unclear. Because the stimulators produced obvious motor effects in humans, or had a comparable peak magnetic field strength, they were assumed to produce comparable electric field intensities and neuronal effects in animal brains. We tested this assumption using 3 stimulus coils of different sizes and design, plus 6 saline-filled spheres that spanned a range of volume from 0.5 to 1800 ml. The induced electric field diminished monotonically with decreasing radius, by factors of 4.7-6.2 at the extremes of size. Comparable results were found using a mathematical model. These results suggest that the efficiency of magnetic stimulation is drastically reduced in smaller brains, and that threshold and safety studies in some animal models may not be valid.


The effects of maturation on spontaneous eye movements in the macaque monkey. The spontaneous eye movements of infant and adult monkeys were studied both in the dark and in the laboratory light by a magnetic search-coil technique and analysed comparatively. The spatio-temporal organization of the infant monkey's eye movements is predominantly vertical; by contrast it is predominantly horizontal in adults. Moreover, the infant monkey's eye movements have smaller amplitudes and slower velocities than adult's in both visual conditions. The linear relationships between amplitude and maximum velocity suggest that rapid eye movements of the infant monkey are saccades but with a lower rate of velocity increase than the adult's. We conclude that the eye movements in the infant and in the adult monkeys differ in many aspects and that maturation acts on both the static and dynamic characteristics of ocular motility.


Abnormal sympathetic skin responses in thalamic lesions. Sympathetic skin responses (SSRs) were abolished in 4 patients affected with fatal familial thalamic degeneration involving the anterior (A) and dorsomedial (DM) thalamic nuclei, without lesions of the peripheral vegetative system. Abnormalities of SSR were not due to peripheral nerve lesions. It is concluded that SSR integrity also depends upon thalamic formations ("visceral" thalamus) and their frontal cortical connections.


Western ligand blot assay for human growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3): the serum levels in patients with classical growth hormone deficiency. The insulin-like growth factors I and II (IGFs), important growth factors both in vivo and in vitro, are known to have at least six binding proteins (IGFBP-1-6). In human serum, IGFBP-3 is a major binding protein and is considered to be GH-IGF-I-dependent. We have established a Western Ligand Blot (WLB) assay for IGFBP-3. The method is a densitometric analysis of IGFBP-3 bands on a film of WLB. The IGFBP-3 levels of patients with classical growth hormone deficiency (GHD, 5 isolated and 10 multiple hormone deficiencies with appropriate therapy) were studied. Before puberty there is no overlap between control (n = 31) and the patients with GHD (n = 10). However, IGFBP-3 levels of two of five pubertal patients with GHD were within the normal range (n = 16). We think that measurement of serum IGFBP-3 is a useful diagnostic marker for GHD, especially before puberty.


Thyroid function before and after induced abortion in normal pregnant women. We assessed thyroid function before and after induced abortion in 25 normal pregnant women. Serum TSH was significantly increased (P less than 0.02), and serum hCG-beta was significantly reduced (P less than 0.001) 1 week after induced abortion, compared with the levels before induced abortion. There was a significant negative correlation between hCG-beta and TSH, and a positive one between hCG-beta and FT4 before induced abortion (P less than 0.02). No difference was observed in thyroid hormones before and 1 week after induced abortion. The results suggest that hCG stimulates the thyroid gland, gaining an advantage over TSH, in normal pregnant women.


Radioimmunoassay for calcitonin gene-related peptide and its measurement in sera of patients with thyroid disease. To investigate serum levels of calcitonin gene-related peptide (CGRP), we developed a sensitive radioimmunoassay (RIA). RIA for CGRP in serum can present some problems: the serum may degradate the tracer during incubation and suppress the antigen-antibody reaction. We avoided these problems by using aprotinin and CGRP-free serum instead of a buffer for the standard curve. We detected serum CGRP in all 39 healthy subjects when CGRP-free serum was not used for the standard curve, but 34 of these subjects had serum CGRP levels below the detection limit (less than 80 pmol/l) when CGRP-free serum was used for the standard curve. We defined the normal range for serum CGRP as below 100.8 pmol/l, which was the maximum level found in the healthy subjects. We studied serum levels of this peptide in patients with thyroid diseases, because the thyroid may be one origin of circulating CGRP. Four of 10 patients with medullary thyroid carcinoma had elevated serum levels of CGRP. Seven of 24 patients with subacute thyroiditis had elevated serum levels of CGRP, but at least one year after clinical recovery, CGRP was undetectable in all. Seven of the 37 patients with hypothyroidism had elevated serum levels of CGRP. None of the patients with hyperthyroidism, adenomatous goiter, thyroid adenoma, or thyroid carcinoma had elevated serum CGRP levels. It is necessary to use a standard curve obtained by the addition of aprotinin and CGRP-free serum to the assay standards to measure serum CGRP levels. Some patients with subacute thyroiditis, hypothyroidism, or medullary thyroid carcinoma had elevated serum CGRP levels.


Production and characteristics of human tissue plasminogen-activator antibodies with region-oriented synthetic peptides. We selected six peptide sequences as belonging to potential epitopes of tissue plasminogen activator (tPA) using, as the main criterion for their choice, the location of the peptide sequences on the surface of the protein molecule. The six peptides (corresponding to amino acids 4-8, 11-16, 96-101, 272-277, 371-376 and 514-519) were synthesized, coupled to carrier proteins and injected into rabbits. All of these peptides elicited antibodies and 15-75% binding of the corresponding iodinated peptide was obtained with a 1:100 dilution of antiserum. Only two anti-(peptide) sera [anti-(tPA96-101) and anti-(tPA272-277)] reacted with intact tPA and its heavy chain in Western immunoblotting analysis. These two peptides sequences and fragment tPA11-16 appear to be involved in the structure of native antigenic epitopes of tPA, since they were recognized and antibodies present in antisera raised against native tPA. There was no interaction between anti-(tPA4-8) and anti-(tPA371-376) sera with intact one-chain or two-chain tPA. In the case of anti-(tPA4-8) cleavage of one-chain tPA to two-chain tPA and reduction of disulfide bonds exposed this epitope.


Substance-P-related and neurokinin-A-related peptides from the brain of the cod and trout. An extract of the brain of the rainbow trout, Oncorhynchus mykiss contained high concentrations of both neurokinin A-like immunoreactivity (corresponding to 90 pmol mammalian neurokinin A/g wet tissue) and substance-P-like immunoreactivity (corresponding to 50 pmol mammalian substance P/g wet tissue) measured by radioimmunoassay using antisera directed against the C-terminal regions of the mammalian peptides. In contrast, an extract of the Atlantic cod. Gadus morhua contained only neurokinin-A-like immunoreactivity (151 pmol/g). This apparent paradox was resolved by determination of the primary structures of the fish tachykinins. Trout substance P (Lys-Pro-Arg-Pro-His-Gln-Phe-Phe-Gly-Leu-MetNH2) has the same amino acid sequence in its C-terminal region as that in the corresponding region of mammalian substance P. Cod substance P (Lys-Pro-Arg-Pro-Gln-Gln-Phe-Ile-Gly-Leu-MetNH2), however, contains a substitution at position 8 (Phe----Ile) that abolishes reactivity with the antiserum to substance P but permits reactivity with the antiserum to neurokinin A. The amino acid sequence of cod and trout neurokinin A is the same (His-Lys-Ile-Asn-Ser-Phe-Val-Gly-Leu-MetNH2) and shows two substitutions (Thr3----Ile and Asp4----Asn) compared with mammalian neurokinin A. The data indicate that nervous tissue of teleost fish contain tachykinins that are analogous to the peptides found in mammalian tissues.


Localization of the cross-linking sites of RGD and KQAGDV peptides to the isolated fibrinogen receptor, the human platelet integrin glycoprotein IIb/IIIa. Influence of peptide length. The non-covalent and Ca(2+)-dependent heterodimer GPIIb/IIIa, formed by platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa), also known as the integrin alpha IIb beta 3, is the inducible receptor for fibrinogen and other adhesive proteins on the surface of activated platelets. A fraction of the isolated GPIIb/IIIa in solution binds RGD or KQAGDV inhibitory peptides and, upon peptide removal, apparently acquires the capacity to bind fibrinogen ('activated' GPIIb/IIIa) [Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C. & Ginsberg, M. H. (1991) Cell 65, 409-416]. Photoaffinity labelling was used here to study the ligand binding site(s) of GPIIb/IIIa in solution, for which the peptides CKRKRKRKRRGDV (alpha 1), CGRGDF (alpha 2), CYHHLGGAKQAGDV (gamma 1) and CGAKQAGDV (gamma 2) were synthesized with a photoactivable cross-linker group and a fluorescent reporter group attached to the N-terminal cysteine residue. Contrary to the situation in activated platelets, both GPIIb and GPIIIa were equally labelled by the four peptides and the cross-linking sites were localized by protein chemical analyses of the fluorescently labelled tryptic peptides of both subunits. Thus, the localization of the cross-linking sites in GPIIb varies considerably with the peptide length and is very different from that localization observed in activated platelets: alpha 2 and gamma 2 were found cross-linked to the N-terminal of both the heavy (GPIIbH 42-73) and the light (GPIIbL2 30-75) chains of GPIIb; while the longer peptides alpha 1 and gamma 1 were cross-linked to the C-terminal of GPIIbH within the 696-724 and 752-768 peptide stretches, respectively. On the other hand, the cross-linking sites of the four inhibitory peptides in GPIIIa were found mainly within the proteolysis susceptible region, between the N-terminal (GPIIIa 1-52) and the core (GPIIb 423-622) highly disulphide-bonded domains, observing that the longer the peptide the closer the cross-linking site is to the N-terminal of GPIIIa: alpha 1 at GPIIIa 63-87 and 303-350; gamma 1 at GPIIIa 9-37; alpha 2 at GPIIIa 151-191; and gamma 2 at GPIIIa 303-350. These results led us to the following conclusions. (a) The GPIIIa 100-400 region contributes to the ligand-binding domain in GPIIb/IIIa both in solution and in activated platelets.(ABSTRACT TRUNCATED AT 400 WORDS)


Purification to homogeneity and characterization of a redoxyendonuclease from calf thymus. A redoxyendonuclease from calf thymus was purified to apparent homogeneity. The redoxyendonuclease recognized and induced cleavage of DNA damaged by ultraviolet light. The enzyme preparation produced a single band of a relative molecular mass of approximately 34 kDa upon SDS/PAGE. The apurinic/apyrimidinic endonuclease and the DNA glycosylase activities remained associated in the apparently homogeneous preparation of the enzyme. The redoxyendonuclease activity displayed a broad pH optimum between pH 5.0-8.5 and exhibited no requirement for divalent cations. By application of FPLC columns Mono-S, Mono-Q and Mono-P, the isoelectric point (pI) of the enzyme was found to be approximately 8.0. Using the DNA sequencing procedure of Maxam and Gilbert [Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560] the purified enzyme was found to incise ultraviolet-light-irradiated DNA at pyrimidine sites as observed previously with a more crude form of the enzyme. While the most frequently cleavaged sites for the crude preparation were at cytosine residues, the apparently homogeneous enzyme preparation frequently induced cleavage sites at both cytosine and guanine residues. Predominant incision induced by the apparently homogeneous preparation was observed at guanine residues when a particular DNA sequence was used as substrate. Furthermore, the 16 N-terminal amino acid residues of the purified enzyme were identified. The sequence did not show any significant similarity to other known proteins.


Comparative study between the use of a treatment with hyperthermia through the rectal approach versus the urethral approach for benign prostatic hyperplasia. 200 patients suffering from benign prostatic hyperplasia were treated with hyperthermia: 100 cases through the rectal approach and 100 through the urethral approach. Subjective symptoms were assessed as well as nycturia and objective data, urinary flow and postmicturition residue before treatment and 6 months after treatment. In the group of 100 patients treated rectally, the subjective symptoms and nycturia improved in 76; urine flow improved in 63, postmicturition residue decreased in 32 and the vesical catheter could be removed in 5 out of 8 patients. With the urethral approach, 77 patients out of 100 presented an improvement in their symptoms, nycturia improved in 53; urine flow improved in 28; the urine residue was decreased in 40 and the vesical catheter could be removed in 10 out of 16 patients who required it previously. Although slightly better results seem to be achieved with the use of rectal hyperthermia, as concerns nycturia and micturition flow, we prefer the urethral approach for its higher degree of convenience, easier handling, shorter time of treatment and reasonable effectiveness.


Differential tissue-specific expression and induction of cytochrome P450IVA1 and acyl-CoA oxidase. We have examined the tissue-specific expression and inducibility of acyl-CoA oxidase and cytochrome P450IVA1 (P450IVA1) RNA in rats. Groups of three rats were dosed daily by gavage with methylclofenapate at 25 mg/kg in 5 ml/kg corn oil for nine weeks, or were administered a vehicle control. P450IVA1 and acyl-CoA oxidase RNA were detected using an RNase protection assay. Similar levels of acyl-CoA oxidase RNA were present in control liver and kidney, but the level of this RNA in lung, muscle and testis was 6-11%, and in pancreas was 0.13%, of that in liver. Treatment of rats with methylclofenapate led to an 11-fold induction of acyl-CoA oxidase RNA in liver and also produced a significant induction of this RNA in kidney, lung, muscle and testis of 1.7-fold, 1.3-fold, 2-fold and 1.7-fold, respectively. Acyl-CoA oxidase RNA was not induced in pancreas. P450IVA1 RNA was present in control liver and also in kidney of control rats at 28% of the level in liver. In contrast to acyl-CoA oxidase RNA, P450IVA1 RNA was not detected in lung, pancreas or testis. Methylclofenapate treatment of rats led to an 18-fold induction of P450IVA1 RNA in liver, and a sevenfold induction in kidney. Induction of P450IVA1 was not detected in any of the other tissues examined. Quantification of the relative amounts of acyl-CoA oxidase and P450IVA1 RNA in control liver revealed that acyl-CoA oxidase RNA was present in a 17.5-fold molar excess over P450IVA1 RNA. Western blotting with an anti-P450IVA IgG revealed two bands of similar apparent molecular mass in liver and kidney microsomes, but not in microsomes from the testis of control rats. Methylclofenapate treatment of rats caused an increase in the intensity of these bands in microsomes from liver, but no induction was obvious in kidney. Immunocytochemical staining for both the microsomal P450IVA and peroxisomal acyl-CoA oxidase proteins was restricted to the proximal convoluted tubule in the kidney cortex, with staining being most intense in the S3 region.


Value of biochemical markers in the management of disseminated prostatic cancer. The biochemical markers alkaline phosphatase (Alk P), prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) were measured 3-monthly in 61 patients with disseminated prostatic cancer who were treated with LHRH analogues. The decrease in Alk P and PSA during the first 6 months of treatment was significantly related to a better survival. In this follow-up study, only PSA was useful for monitoring prostatic cancer during hormonal treatment. Before it was visible on a bone scan, PSA gave an indication of tumor progression. PSA might permit omission of routine bone scanning. Consensus must be obtained about the cost-saving use of biochemical markers in the treatment of disseminated prostatic cancer. With the number of treatment options increasing, objective measures are of utmost importance. Biochemical markers can be used for prognosis and monitoring of the treatment of patients with disseminated prostatic cancer.


Advantage of systematic random ultrasound-guided biopsies, measurement of serum prostate-specific antigen level and determination of prostate volume in the early diagnosis of prostate cancer. Two hundred and sixteen patients, presenting with a suspicious digital examination (stage T3 excluded) or a level of prostate-specific antigen (PSA) greater than or equal to 2.5 ng/ml, assessed by radioimmunoassay, underwent a transrectal ultrasound examination. Prostate volume was systematically calculated and correlated to PSA level. Biopsies were performed: (1) on suspicious peripheral hypoechoic areas; ultrasound-guided biopsies; (2) systematically on the 2 prostate lobes, whatever the result of transrectal ultrasound imaging:random systematic ultrasound-guided biopsies. In the 186 patients who had never undergone prostate surgery, ultrasound-guided biopsies showed 42 prostate cancers and random systematic ultrasound-guided biopsies showed 75; 14 of the 76 patients with normal digital rectal examination and transrectal ultrasound imaging had a prostate cancer. In the 30 patients who had previously undergone surgery for benign prostatic hypertrophy, random systematic ultrasound-guided biopsies showed 18 prostate cancers, 13% more than ultrasound-guided biopsies; 75% of patients with a serum PSA greater than 5 ng/ml had a prostate cancer. A very significant correlation was found between PSA level and prostatic volume (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of neutral endopeptidase inhibitor on airway function and bronchial responsiveness in asthmatic subjects. We determined the effect of an inhibitor of neutral endopeptidase, acetorphan, on the skin responses to substance P and on the bronchostrictor effects of sodium metabisulphite aerosol in asthmatic subjects. One hour following ingestion of acetorphan (200 mg) or placebo tablets, cutaneous responses to substance P were performed in four subjects. In seven subjects, bronchial challenge with increasing concentrations of sodium metabisulphite solutions was performed and the concentration required to cause a 20% fall in baseline FEV1 determined (PC20). On the acetorphan day, there was a significant increase in the wheal and flare responses to substance P and to the diluent (0.9% NaCl) alone. However, there was no significant effect of acetorphan on the PC20 metabisulphite. We conclude that metabisulphite airway challenge in vivo may not invoke the release of endogenous neuropeptides. However, the degree of inhibition of neuropeptide breakdown by the oral dose of acetorphan used may not have been optimal.


The A2 isoform of rat Na+,K(+)-adenosine triphosphatase is active and exhibits high ouabain affinity when expressed in transfected fibroblasts. The alpha isoforms of the Na+,K(+)-ATPase (Na+ pump) are expressed with developmental and tissue heterogeneity in rodents and possess different sensitivity to inhibition by ouabain. We directly characterized the ouabain sensitivity of the rat A2 (alpha 2) isoform by transfecting NIH 3T3 cells with rat A2. The treated cells exhibit high affinity (40 nM) ouabain binding with a density of 2 pmol/mg protein. 86Rb+ flux studies confirm that A2 is functional in this system and that A2 is inhibited by submicromolar concentrations of ouabain. These findings are consistent with measurements of ouabain affinity in tissues which express the A2 isoform.


Differential regulation of insulin-like growth factor binding protein (IGFBP)-2 mRNA in liver and bone cells by insulin and retinoic acid in vitro. Isolated cells produce insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). Two distinct cell types were studied with regard to IGFBP-2 expression: (i) rat hepatocytes, which produce IGF I at a high rate and thus regulate its plasma concentration; and (ii) rat osteoblasts, which are targets of IGF I action. IGFBP-2 expression is low in hepatocytes prepared from normal adult rats and high in calvaria cells from newborn rats. Retinoic acid stimulates IGFBP-2 production by liver cells. Insulin suppresses both basal and retinoic acid-induced IGFBP-2 mRNA expression in hepatocytes and has no such effect on osteoblasts. Retinoic acid and insulin regulate IGFBP-2 expression in a tissue-specific manner.


Bleomycin-detectable iron in the plasma of premature and full-term neonates. The bleomycin assay measures non-transferrin-bound iron, able to catalyze free radical reactions, in human plasma. No bleomycin-detectable iron is present in plasma from healthy adults. However, plasma from 3/15 premature babies was positive in this assay. Plasma from 52 apparently-healthy term babies was analyzed and 11 were positive in the bleomycin assay. Hence not only some premature but also some full-term apparently-healthy babies may be at risk of severe oxidative damage.


Do antigenic peptides have a unique sense of direction inside the MHC binding groove? A molecular modelling study. In the models suggested recently for antigenic peptides binding in the alpha 1, alpha 2 groove of MHC class I molecules, the orientation of the peptide has been shown uniquely as: the N----C vector of the peptide being parallel to the N----C vector of the alpha 1 helix of MHC. Here, we demonstrate that the reverse orientation of the peptide is equally probable. This hypothesis is supported by molecular modelling calculations and computer graphic analyses on a murine class I MHC molecule H-2Kd and its complexes with a restricted peptide RYLENGKETLQ. Analysis of the complementary interactions between the peptide residues and the amino acid side chains lining the MHC groove shows that the binding orientation of the peptide may be allele-specific and could depend on the sequence and structure of the antigenic epitope.


DNA fingerprints of Bombyx mori L. Testing of genotypic variability of parthenogenetic strains. A comparative analysis of the total cellular DNA in certain parthenogenetic specimens of the silkworm (Bombyx mori), produced from the females of the parthenogenetic strains by two types of parthenogenesis, has been performed through the application of the DNA fingerprinting method based on M13 phage DNA as a hybridization probe. It has been shown that parental specimens and their genetically identical off-springs produced through ameiotic parthenogenesis have identical patterns of hybridization with the hypervariable DNA fragments. The off-springs produced through the meiotic type of parthenogenesis have individual-specific patterns of hybridization, revealing a high level of polymorphism of individual genotypes. The results obtained testify to the effectiveness and reliability of this promising method for identification of genotypic variability, marking and genomic characterization of parthenogenetic clones in the silkworm.


Zinc causes tyrosine phosphorylation of hippocampal p60c-src. Zinc cations at concentrations of 0.2 mM and greater catalyzed specific phosphorylation, by ATP, of two membrane-associated proteins from rat hippocampus. These proteins, corresponding to molecular weights of 60 and 49 kDa, were phosphorylated primarily at tyrosine residues. The 60-kDa protein was identified as p60c-src by immunoprecipitation using two different p60src-specific monoclonal antibodies. The 49-kDa protein co-immunoprecipitated with p60c-src. Cyanogen bromide cleavage of p60c-src and the 49-kDa protein phosphorylated in the presence of Zn2+ gave different patterns of phosphopeptides. It is suggested that tyrosine phosphorylation of p60c-src and the p60c-src-associated 49-kDa protein may be a way of zinc participation in hippocampal neurotransmission.


Cryosurgery for locally recurrent rectal cancer. Locally recurrent rectal cancer is, in most cases, unresectable and incurable. Palliative treatment is warranted in many cases because of the presence of severe distressing symptoms. In recurrent disease, intraluminal cryotherapy is an option for palliation. Twenty patients with local recurrence after anterior resection were treated palliatively with cryosurgery for their local symptoms. Six patients had previously had a colostomy before they were referred for palliative treatment. Thirteen patients had more than one symptom. Distant metastases were present in ten cases. The beneficial effect of cryosurgery was evident after two to three sessions. In nine patients cryotherapy achieved complete relief of local symptoms. In these patients the symptom free interval varied from 1 to 24 months (median 11 months); five patients died of disease without local symptoms. Three of these nine patients underwent a bowel diversion at a later stage because of complete stenosis. The number of treatment sessions in this group of patients varied from three to 14. The palliative index varied from 37 to 100% (mean 78%). In nine patients cryotherapy of the local recurrence gave no relief at all. Our results show that in almost half of the patients cryosurgery can palliate local complaints resulting from recurrent tumor growth after anterior resection.


Anti-CD3 immunotoxin prevents low-dose STZ/interferon-induced autoimmune diabetes in mouse. Autoimmune diabetes was induced with an established model in which 3 daily injections of 95 mg/kg body wt/day streptozocin (STZ) and 2 x 10(4) U interferon-gamma (IFN-gamma) were administered to C57BL/6 mice. Diabetes onset was accompanied by precipitous increases in serum glucose levels and validated by immunoperoxidase studies showing diminished islets in pancreatic tissue sections. Administration of two to three doses of a monoclonal antibody (MoAb) or an immunotoxin (IT) directed against the CD3 epsilon-chain before STZ/IFN-gamma treatment prevented increases in serum glucose and protected islets from damage. IT was made by crosslinking anti-CD3 to a low oligosaccharide-containing fraction of purified ricin toxin A chain (RTA; a catalytic inhibitor of protein synthesis) with a stabilized derivative of 2-iminothiolane. Protection was complete, long-lived, and selective because two different control ITs did not prevent diabetes onset. A second pan T-cell-reactive IT was synthesized by linking the MoAb anti-Ly1 to the same RTA toxin. Anti-Ly1 reacts with the murine homologue of human CD5. Anti-Ly1 RTA also protected against diabetes onset in a dose-dependent manner requiring higher doses and a longer schedule than anti-CD3 or anti-CD3 RTA. These studies demonstrate for the first time the importance of CD3+ and CD5+ cells in diabetes onset in the low-dose STZ/IFN-gamma model and show that anti-CD3, anti-CD3 RTA, or anti-CD5 RTA may be useful in vivo for the treatment of diabetes or perhaps other T-cell-mediated autoimmune diseases. These data may have important therapeutic implications for early autoimmune diabetes in humans.


Focal induction of IGF binding proteins in proximal tubules of diabetic rat kidney. Diabetes-associated kidney enlargement is associated with increased kidney insulinlike growth factor I (IGF-I) binding. IGF-I binds to the type I IGF receptor, which mediates most of its actions, and to specific binding proteins (IGFBPs), which modulate its actions. To explore the nature and extent of IGF-I binding in the kidney, in vitro autoradiography was used to map the distribution of IGF binding in control and diabetic rat kidney. Specificity studies were performed with increasing concentrations of unlabeled IGF-I, IGF-II, des(1-3)IGF-I (an IGF-I derivative that binds to receptors normally but with decreased affinity to binding proteins), and insulin. In control rats, diffuse binding was found throughout the kidney with increased density in the papilla. Binding specificity in the cortex and outer medulla was typical of the type I IGF receptor (IGF-I = des[1-3]IGF-I greater than IGF-II much greater than insulin). Binding in the outer medulla of diabetic kidney was typical of the type I IGF receptor. A marked focal increase in proximal tubular binding occurred in 13 of 22 postpubertal diabetic rats. Binding specificity of the proximal tubular binding was consistent with the predominance of an IGF binding protein (IGF-I = IGF-II greater than des[1-3]IGF-I with minimal displacement by insulin). Northern-blot analysis revealed increased IGFBP-1 and IGFBP-3 mRNA in cortical tissue from diabetic rats displaying increased proximal tubular binding but not from diabetic rats not displaying this phenomenon. As cell surface association of IGFBPs is linked to potentiation of IGF activity, a possible mechanism for potentiation of local IGF-I action may be provided.


Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction. Increased blood flow and vascular leakage of proteins preferentially affect tissues that are sites of diabetic complications in humans and animals. These vascular changes in diabetic rats are largely prevented by aminoguanidine. Glucose-induced vascular changes in nondiabetic rats are also prevented by aminoguanidine and by NG-monomethyl-L-arginine (NMMA), an established inhibitor of nitric oxide (NO.) formation from L-arginine. Aminoguanidine and NMMA are equipotent inhibitors of interleukin-1 beta-induced 1) nitrite formation (an oxidation product of NO.) and cGMP accumulation by the rat beta-cell insulinoma cell line RINm5F, and 2) inhibition of glucose-stimulated insulin secretion and formation of iron-nitrosyl complexes by islets of Langerhans. In contrast, NMMA is approximately 40 times more potent than aminoquanidine in elevating blood pressure in nondiabetic rats. These results demonstrate that aminoguanidine inhibits NO. production and suggest a role for NO. in the pathogenesis of diabetic vascular complications.


[Palliative laser therapy in gynecologic oncology]. Local recurrences of carcinomas of the breast and genitals are a severe psychic and physical strain on the patients. Continuous confrontation with an often visible and generally painful tumour manifestation is an indication for palliative treatment, if other oncological therapies can no longer be employed. In these cases, the possibility of laser use represents a new therapeutic approach. In a pilot study at the University of Heidelberg, Department of Obstetrics and Gynaecology, a palliative laser therapy was performed on 45 patients with locally recurrent carcinomas of the breast (n = 29) and genitals (n = 16). Carbon dioxide and Nd:YAG lasers were utilized for tumour resection, vaporisation and coagulation. This new concept, the combined application of both wavelengths has been proved to be most efficient.


Pancreatitis following bile duct sphincter of Oddi manometry: utility of the aspirating catheter. The aspirating sphincter of Oddi manometry (SOM) catheter was shown to reduce the frequency of post-procedure pancreatitis from 31% to 4% following a pancreatic duct evaluation. This study was designed to prospectively evaluate the utility of the aspirating manometry catheter in reducing the frequency of pancreatic enzyme elevation and clinical pancreatitis following isolated bile duct manometry. Thirty-eight patients were randomly assigned to undergo bile duct SOM with the standard perfusion (infused group) catheter or the aspirating catheter (aspirated group). Overall, the frequency of both amylase and lipase level elevation at least two times the upper limits of normal was 30% at 2 hours, 25% at 6 hours, and 18% at 18 hours after the procedure and was similar for the aspirated and infused groups. No episodes of clinical pancreatitis occurred in either group. The SOM catheter was perfused with full-strength contrast in 12 consecutive patients undergoing a bile duct evaluation. Only one patient had any contrast material identified in the pancreatic duct. The results of this study support the theory that increased pancreatic duct hydrostatic pressure is the major cause for post-SOM pancreatitis and suggests that SOM evaluation of the bile duct alone appears to be safe.


Palliation by laser photoablation: a multidisciplinary quality assessment. To evaluate the quality of life and degree of palliation by laser photoablation for gastrointestinal cancer, a questionnaire was sent to general practitioners (GPs) and referring specialists. The response was 85%. General practitioners considered palliation by laser to be effective in 74% compared with 50% for referring specialists (p less than 0.001). Specialists felt themselves unable to answer in 17% compared with no GPs (p less than 0.001). The differences in assessment between specialists and GPs were most pronounced in colorectal cancers. There was a striking consensus of opinion about the rate of failed palliation among endoscopist, referring specialist, and GP. On the other hand, the rating of success by specialists and GPs was significantly lower than the endoscopist's evaluation. The endoscopist and GP were more at variance than the endoscopist and specialist. Above all, the GPs seemed to outweigh the burden against the benefits of treatment. Disagreement of the specialists and the GPs with the endoscopist about the outcome appeared to be related to unrealistic expectations, to a shift in presenting symptoms or to complications, misinterpreted as being laser-induced but mainly due to progression of disease.


[Effect of tranilast, an anti-allergic drug, on the human keloid tissues]. We studied the inhibitory effects of tranilast, an anti-allergic drug, on the human keloid tissues implanted into the dorsal skin of athymic nude mice and on the growth of keloid fibroblast in vitro. In the keloid tissue-implanted model, tranilast (50-200 mg/kg, p.o.) decreased the weight of the keloid tissue as triamcinolone (25 mg/kg, p.o.) did. Tranilast (200 mg/kg, p.o.) reduced the hydroxyproline content of implanted tissues. Tranilast (3-300 microM) also inhibited the collagen synthesis by keloid fibroblast in vitro. Only a high concentration of tranilast (300 microM) suppressed the glycosaminoglycan synthesis and cell proliferation of keloid fibroblasts. Moreover, tranilast scarcely affected the fibronectin production. Triamcinolone (10 microM) also inhibited glycosaminoglycan synthesis and cell proliferation. These results suggest that the inhibitory effect of tranilast on the keloid tissues is related to its inhibition of the collagen synthesis of fibroblasts. Tranilast would be useful as a therapeutic drug for the treatment of keloids.


[Effect of tranilast, an anti-allergic drug, on carrageenin-induced granulation and capillary permeability in rats]. We studied the effect of tranilast on the growth of carrageenin-induced granulation and the increase in capillary permeability induced by inflammatory agents in rats. In the carrageenin-induced granulation model, tranilast (50 or 100-200 mg/kg, p.o.) decreased significantly and dose-dependently the weight and the hydroxyproline content of the granulation tissue. Tranilast, however, showed no effect on the healing day of locally wounded dorsal skin of rats. Triamcinolone (10 mg/kg, p.o.) also showed an inhibitory effect on the carrageenin-induced granulation model. Tranilast (50-400 mg/kg, p.o.) dose-dependently inhibited the enhancement of capillary permeability induced by the Ca ionophore A23187, bradykinin and xanthine oxidase. Moreover, tranilast (30 and 300 microM) suppressed superoxide production induced by FMLP in human neutrophils, but did not act as a superoxide scavenger. Considering that hypertrophic scar and keloid are conditions characterized by abnormal cell proliferation and excessive collagen accumulation accompanied with itch and pain, these results suggest that tranilast is useful as a therapeutic drug for hypertrophic scars and keloids.


Comparative effect of truncal vagotomy and pirenzepine on postprandial secretory response of exocrine pancreas in dogs. The comparative effect of truncal vagotomy and associated pyloroplasty and orally administrated pirenzepine (75 mg, 1 h before food intake) on the exocrine pancreatic secretory response to food was studied in conscious dogs. Both pirenzepine administration and truncal vagotomy totally abolished the pancreatic hydromineral and organic secretory response to food intake while postprandial pH of intraduodenal content remained above 6. From the results of our study we conclude that the vago nerve must not be altered in order to assure a pancreatic response to food intake and that pirenzepine is as strong as truncal vagotomy in maintaining pH in postprandial intraduodenal content over 6.


Sclerosing adenosis of the prostate. Report of three cases with electronmicroscopy and immunohistochemical study. Three cases of rare variant of prostatic adenosis with the features of sclerosing adenosis, an uncommon lesion sometimes confused with prostatic carcinoma, are reported. The lesions consisted of a small, single nodule in prostates otherwise showing typical adenomatous and fibromuscular hyperplasia. The lesions were composed of crowded small glands, small solid nests and individual cells embedded in a cellular stroma. Immunohistochemistry showed that the glands were lined by basal cells positive for the high-weight keratins (EAB-903 and AE-3), a finding which confirms the benign nature of the lesion. S-100 protein and smooth muscle actin were also positive in the same basal cells suggesting myoepithelial differentiation, a character not found in basal cells outside this lesion. This finding was confirmed at ultrastructural level by the finding of numerous thin filaments in the cytoplasm of basal cells. It is important to recognize this lesion in order to avoid confusion with well-differentiated prostatic carcinoma.


Effects of tetrahydro-9-aminoacridine on the electrocorticogram of rats with a unilateral lesion of the nucleus basalis magnocellularis. The effects of tetrahydro-9-aminoacridine (THA) were studied on the electrocortical activity of control rats and of rats with a unilateral lesion of the nucleus basalis magnocellularis (NBM) produced by alpha-amino-3-hydroxy-4-isoxozole propionic acid (AMPA). This lesion almost completely deprived the lesioned hemisphere of its cholinergic innervation. In control rats, THA (10 mg/kg i.p.) increased the amplitude of the slow components of the electrocorticogram (less than 9 Hz). These effects were antagonised by atropine (5 mg/kg i.p.). Lesion of the NBM alone decreased the amplitude of frequencies in the 12-16 Hz frequency band but did not significantly affect the slower frequencies. THA (10 mg/kg) restored the amplitude of the 12-16 Hz activity to the level seen in control rats before THA but did not affect activity in the other frequency bands. The results suggest that THA requires some residual cholinergic innervation in order to exert its effect.


A single human monoclonal antibody that confers total protection from tetanus. Protective human monoclonal antibodies (HuMAbs) are superior to hyperimmune sera and murine monoclonal antibodies as far as human immunotherapy is concerned. In this report, we describe the successful generation of triomas secreting HuMAbs to tetanus toxin (tt). Lymphoblastoid cell lines secreting anti-tt antibodies were stabilized by back-fusion with a mouse x human heterohybrid myeloma partner, SBC-H20. One of the antibodies so produced, confers total protection of mice from tetanus, unlike a few recent reports where only partial protection (delay in the onset of tetanus) was achieved with single HuMAbs. Experiments to localize the neutralizing epitope(s) of the toxin using the protective monoclonal antibodies revealed that the antibody recognizes a conformational determinant that is destroyed on SDS-treatment. Preliminary studies show that Fab preparations of the protective antibody are capable of neutralizing tetanus toxin, suggesting that it might be possible to clone and express the Fab in a stable vector for large scale production.


Identification of an epitope recognized by the monoclonal antibody PEP80 in the C-terminal cytoplasmic fragment of glycophorin A. The monoclonal antibody PEP80 (IgG1) was raised by immunization of BALB/c mice with asialo-agalacto-glycophorin from human erythrocytes. The antibody is specific for glycophorin A (GPA) and reacts strongly with the GPA-derived tryptic peptide which is the C-terminal cytoplasmic portion of GPA, containing amino acid residues 102-131. Using the smaller chymotryptic fragments of this peptide and a set of solid phase-synthesized peptides allowed to establish that the MAb PEP80 is directed against an epitope comprising amino acid residues 112-121 of GPA. The peptides terminated with 120th or 119th amino acid residue were slightly less active, and the minimal structure which still gave a weak reaction with the antibody was the sequence of amino acid residues 112-118. The MAb PEP80 did not bind to live human erythroleukemic K562 cells, but showed a strong binding to the cells permeabilized with methanol.


High level expression in E. coli of an alternate reading frame of pS2 mRNA that encodes a mimotope of human breast epithelial mucin tandem repeat. The high molecular weight mucin found in human milk fat globule and on the surface of mammary and other epithelial cells contains a 20 amino acid tandem repeat sequence that is highly immunogenic. We have immunoscreened lambda gt11 cDNA expression libraries from MCF7 cells and lactating breast tissue with 5 anti-mucin monoclonal antibodies. We isolated a group of cDNA clones that had the repeat sequence (HB11-2, HB11-6, HB11-10) and a group that had little or no homology with the repeat sequence (NP4, NP5, HB11-4). A fusion protein produced by NP4 bound preferentially BrE2, while HB11-4 bound only BrE2 and BrE3, NP5 produced a fusion protein that bound only Mc5 and not the other MAbs. Sequencing of the NP5 cDNA revealed it to be distinct from the mucin sequence and instead to have 97% identity with the estrogen induced transcript, pS2. An alternate reading frame was translated by the lambda gt11 fusion gene yielding a 44 amino acid protein having no homology with pS2 protein. Only a short region of homology (5 amino acids) with the breast mucin tandem repeat was found which was shown to be a mimotope for the Mc5 epitope on the breast mucin. High level expression of the NP5 cDNA was achieved by subcloning it into pEX2. The NP5 fusion protein has been useful for developing an assay for the presence of mucin derived antigen in patient serum.


Production and characterization of two new mouse monoclonal antibodies reactive with denatured mouse class II beta chains. We report on the production and characterization of two murine peptide specific anti-major histocompatibility complex class II chain specific monoclonal antibodies. Two new mouse monoclonal antibodies reactive with two synthetic peptides corresponding to Abb amino acids (146-157) and Abs amino acids (146-157) were produced. KL295 is the mouse anti-Abb monoclonal antibody, which reacts with denatured beta chains of H-2b, H-2d, H-2p, and H-2q, but fail to react with spleen cell lysates from H-2f, H-2j, H-2k and H-2s mice. The mouse anti Abs monoclonal antibody, KL304 in contrast reacts with the denatured Class II beta chains of H-2f, H-2j, H-2k and H-2s mice, but fails to bind H-2b, H-2d, H-2p or H-2q beta chain, suggesting residues 153-155 of this molecule to be critical for the epitope. Both KL295 and KL304 bind B10.WB (H-2j) which suggests a unique epitope in this strain of mice. Neither KL295 or KL304 react with native mouse class II cell surface molecules.


Monoclonal antibodies against bacterial glucosamine 6-phosphate synthase: production and use for structural studies. Fifteen mouse x rat hybridoma cell lines producing rat monoclonal antibodies (MAbs) directed to Escherichia coli Glucosamine 6-P Synthase (GlmS) were established and characterized. Most of them (13/15) are IgG2a while 2 were typed as IgG1. Their Kaff ranged from 1.5 x 10(6) to 9.6 x 10(8) M-1 as determined by Beatty et al. (1). The epitopes recognized by these MAbs were assigned to one of the two catalytical domains of the enzyme (CT1 and CT2) as demonstrated both by ELISA and Western-blotting using purified GlmS proteolytic fragments. The binding of the MAbs on either the native or denatured forms of GlmS, CT1 and CT2 was further analyzed by competitive immunoassay and most of the MAbs were found to bind preferentially to the denatured proteins. The study of the antigenic topography of GlmS by competitive radioimmunoassay demonstrated the existence of at least 10 independent epitopes on GlmS, divided into three groups. The first one (3/15) includes MAbs whose binding was not inhibited by any of the other MAbs. The second group (9/15) is comprised of MAbs that exhibit reciprocal binding inhibitory activity while the third group includes MAbs (3/15) presenting asymmetric inhibitory activity. Finally, since most of the isolated antibodies (10/15) bind to the 27 kDa amino-terminal glutamine binding domain (CT2), the capacity of these MAb to interfere with the associated glutaminase activity was analyzed.


Characterization of the colorectal antigen IOR-C2. A colorectal antigen (IOR-C2) was characterized by a monoclonal antibody produced against the colon cancer cell line SW1116. By immunohistochemical staining the antigen was abundant and strongly expressed in epithelium of normal colon whereas colorectal carcinomas showed a more variable and heterogenous reactivity to the antibody (IOR-C2). Radioimmunoprecipitates of SW1116 cell homogenates showed a 160-200 kD band in SDS gels. Physicochemical characterization indicate that at least two IOR-C2 reactive sites are present on the antigen tested and that it is mainly an 0-linked glycoprotein carbohydrate chain which can also be N-linked to the protein. The expression of IOR-C2 mimics that of the colon associated antigen (CAA) and NCC-CO-450 antigen but is distinct from these with regard to its expression in carcinomas as well as its physicochemical characteristics.


Homologous and heterologous adenylate cyclase system desensitization in glial cells. In the present research the desensitization of adenylate cyclase system induced by isoproterenol (IPR), a beta-adrenergic agonist, in primary glial cell cultures and the effects of an exposure to 3-isobutyl-1-methylxanthine (IBMX) on the response to a subsequent stimulation with IPR have been investigated. A pretreatment with the phosphodiesterase inhibitor IBMX induced refractoriness to a subsequent IPR challenge suggesting a possible involvement of cAMP in heterologous desensitization. Moreover the present results show that IPR desensitized cells in confluent cultures retained a normal response to cholera toxin, while IBMX treated cells exhibited a reduced response to the toxin. So IPR induces a rather specific desensitization while IBMX induced refractoriness seems to be non specific.


Pattern-reversal electroretinographic acuity in untreated eyes with subfoveal neovascular membranes. To define further the natural history of visual loss in eyes with age-related macular degeneration (ARMD) complicated by a subfoveal neovascular membrane, pattern-reversal electroretinograms (ERGs) were obtained from patients randomized to no treatment at the Dallas center of the Macular Photocoagulation Study (Texas Retina Associates). Study eyes (n = 20) were tested during the initial visit and at 3-, 6-, and 12-month follow-up visits. Responses were obtained to phase-alternating checkerboards of varying check size. Extrapolation of the best-fit regression line relating the logarithm of the check size to amplitude was used to determine "retinal" acuity (log MAR). The pattern-reversal ERG acuity rating (100 - [50 x log MAR]) was derived for each visit. Pattern-reversal ERG acuity ratings for all patients across visits were correlated significantly with visual acuity ratings derived from the Bailey-Lovie chart (r = 0.61, P less than 0.001) and inversely related to neovascular membrane area (r = -0.55, P less than 0.001). During 1 yr of follow-up, pattern-reversal acuity ratings dropped from 53 to 12, corresponding to an average decrease of approximately 0.2 octaves/month. These results suggest that the pattern-reversal ERG, which samples the resolving power of the central 20 degrees, is a sensitive index of visual loss in age-related macular degeneration.


Phase II trial of fazarabine in advanced colorectal carcinoma. A total of 15 patients with measurable advanced colorectal adenocarcinoma were prospectively treated with fazarabine (Ara-AC), reconstituted in dimethyl sulfoxide, and administered at a starting dose of 48 mg/m2/day as a continuous intravenous infusion for three days. The dose was repeated every 21 days and dose escalations or reductions were made on the basis of toxicities encountered in the preceding course. No patient achieved either a complete or partial response. Major toxicities encountered were granulocytopenia, thrombocytopenia, nausea, vomiting, anemia, and headache. All toxicities were reversible upon discontinuation of the drug and no life-threatening toxicities occurred. These data indicate that further clinical trials in colorectal carcinoma with this agent and schedule of administration are not warranted.


Phase II trial of fazarabine (ARA-AC, arabinosyl-5-azacytosine) in metastatic breast cancer. Fourteen consecutive female patients with metastatic breast cancer received a total of 31 courses of fazarabine at a dose of 2 mg/m2/hour x 72 hours (48 mg/m2/day x 3) as a continuous infusion. There were no responses seen, although one patient achieved stability of her disease for five courses. Because of encouraging in vitro results and the primarily hematologic dose-limiting toxicity, further study in combination with colony stimulating growth factors might be of interest.


Magnetic resonance imaging of abscesses using lipid-coated iron oxide particles. The authors investigated whether iron oxide particles can be used as a magnetic resonance imaging (MRI) contrast agent to image abscesses in a two-stage experimental design. Human buffy coat was incubated with iron oxide particles of different sizes and coatings. Smears of the incubation mixture were made on a glass slide and stained for iron. The percentage of iron oxide uptake was determined by counting 100 neutrophils and monocytes and scoring the number of cells that contain iron. Subcutaneous abscesses were created in the flanks of 18 Sprague-Dawley rats by injecting them with 0.1 mL of turpentine. Iron oxide was given intravenously, and the animals were imaged by MRI (1.5 T) 12 to 24 hours later. Different iron oxide coatings and doses were compared. The four different types of coating (constant fragment [Fc] of IgG, bovine serum albumin [BSA], lipid [Ferrosome], and dextran) had an uptake of 72% +/- 5.3%, 61% +/- 6.2%, 30.5% +/- 6.8%, and 5% +/- 2.5%, respectively. Comparison of two particle sizes (mean, 90 versus 35 nm) showed the large particles to have higher uptake (61% +/- 6.2%) compared with the small particles (6% +/- 1.8%) (P less than .001). Post-contrast imaging of the rats showed a hypointense ring around the abscess only in the animals injected with the lipid-coated agent. The effect was discernible within 12 hours after contrast injection and at a dose of 25 mumols iron/kg. Histologic sections showed phagocytic cells with iron granules in the periphery of the abscess. No hypointense ring on MRI or iron granules on histologic sections was seen around the abscess of the control animals or those injected with BSA-iron oxide or Fc-iron oxide. Lipid-coated iron oxide particles can be used to image abscesses by virtue of their phagocytosis into surrounding inflammatory cells. Positive uptake of these particles by human phagocytes in vitro suggests that similar results may be applicable in humans.


Detection and quantitation in rat tissues of the superparamagnetic magnetic resonance contrast agent dextran magnetite as demonstrated by electron spin resonance spectroscopy. The compound studies in this article is a superparamagnetic macromolecular complex of magnetite cores coated with hydrophilic dextran, which is under active investigation as a contrast agent for magnetic resonance imaging (MRI) in liver and spleen. The biodistribution of paramagnetic compounds is problematic and is usually studied by histochemical reactions or by radiolabeling the compound under study. The purpose of this article is to show how electron spin resonance (ESR) spectroscopy detects dextran magnetite (DM) particles in tissues. DM injected intravenously in the experimental animal was detected in some reticulo-endothelial organs by ESR. The spectroscopic study was validated using electron microscopy and electron-probe microanalysis. DM exhibits an ESR spectrum; ESR delineated the distribution of DM distribution in liver, spleen, bone marrow, and blood as a function of time. The blood clearance was biphasic, dependent on the size of particles. ESR spectroscopy is a highly sensitive and reproducible method of studying DM distribution.


DNA double labelling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication. A procedure was developed that very effectively distinguishes between IdUrd and CldUrd incorporated in the DNA of cell nuclei and chromosomes. For double staining we used the rat anti-BrdUrd monoclonal antibody from Sera-lab that binds specifically to CldUrd and BrdUrd but not to IdUrd, in combination with the mouse anti-BrdUrd monoclonal antibody from Becton Dickinson. This antibody binds to all three halogenated deoxyuridines, but when the nuclei are washed in TRIS buffer with a high salt concentration the antibodies linked to CldUrd-labelled DNA are removed. When analysing the effect of the deoxyuridines on the cell cycle we found that the growth kinetics of Chinese hamster cells were not changed by adding IdUrd or CldUrd for 30 min at a concentration of 10 microM, whereas adequate double labelling required only 2 min pulses. The effectiveness of the technique was demonstrated in two model experiments. The first test concerned the assessment of cell recruitment in the central areas of slow-growing clones, after addition of fresh medium. The second experiment focussed on the spatial resolution of the method. Double-labelled metaphase chromosomes showed interspersed green and red replication bands with a spacing corresponding with medium resolution Giemsa banding patterns.


Distribution of taurine in the rat cerebellum and insect brain: application of a new antiserum against carbodiimide-conjugated taurine. The production, specificity and application of an antiserum against taurine conjugated to succinylated ovalbumin by means of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide is reported. The antiserum was produced in rabbits. The carbodiimide was used also as a tissue fixative. The development of the antibody titre was followed by dot-blot tests on nitrocellulose filters using different amino acid conjugates and with immunohistochemical reaction in the rat and insect brain. Blocking controls were also used. Taurine antiserum, sufficiently specific and sensitive, developed after the fourth booster injection, after which the antiserum was characterized. In the insect brain, intense taurine-like immunoreactivity was observed in the photoreceptors, in the Kenyon cells and the neuropile of the mushroom bodies, in the lower part of the central body and in the antennal lobes. In the rat carebellum, intense taurine-like immunoreactivity was seen in the Purkinje cells. Immunoreaction was seen also in small cells most probably corresponding to the basket cells. The use of the carbodiimide in the production of antisera against taurine provides a parallel method for comparison of the distribution of taurine-like immunoreactivity obtained with antisera made against conjugates prepared with aldehydes.


The distribution of calcium in undecalcified bone as revealed by an improved pyro-antimonate method. The localization of pyro-antimonate-precipitable Ca2+ in the undecalcified femur and calvaria of neonatal rats was examined. The fixation of bones with pyro-antimonate-glutaraldehyde followed by pyro-antimonate-osmium (two-step method) resulted in better preservation of tissue and more precise localization of precipitates than did the direct immersion of specimens in pyro-antimonate-osmium solution (one-step method). The precipitate was frequently observed within the endoplasmic reticulum of obsteoblasts. Most vacuoles in osteoclasts contained precipitate. By contrast, the mitochondria in these cells were associated with small amounts of precipitate. There was no evidence of precipitate in the Golgi apparatus. The presence of calcium in the precipitate was verified by EGTA treatment and X-ray microanalysis. This study demonstrated that (1) the two-step pyro-antimonate method is a useful and reliable procedure for visualizing Ca2+, and (2) cellular Ca2- can be successfully localized in undecalcified bone by this method.


Study of prostatic disease in dogs: 177 cases (1981-1986). Historical and physical signs associated with prostatic disease diagnosed in dogs over a 5.5-year period were defined. One hundred seventy-seven male dogs were determined to have prostatic abnormality. Of the 177 dogs, 87 were determined to have specific prostatic disease. The most common prostatic disease identified in this study was bacterial prostatitis, followed by prostatic cyst, prostatic adenocarcinoma, and benign hyperplasia. The most common prostatic disease identified in neutered dogs was prostatic adenocarcinoma. Mean age at onset of prostatic disease was 8.9 years; statistically significant difference was not observed between age at onset of the various types of prostatic disease identified. Doberman Pinscher was the most common breed with prostate disease. Twenty-nine percent of dogs with a specifically identifiable prostatic disease had signs of systemic illness, 41% had signs of lower urinary tract disease, 28% had signs of gastrointestinal tract abnormalities, and 13% had signs of locomotor difficulty.


Role of serum vitronectin and fibronectin in adhesion of fibroblasts following seeding onto tissue culture polystyrene. The suitability of polymeric biomaterials as surfaces for the attachment and growth of cells has often been investigated in tissue culture. In this study the contribution that adsorption of serum fibronectin (Fn) or vitronectin (Vn) make to the attachment and spreading of fibroblast cells during the first 90 min following seeding was determined for two modified tissue culture polystyrenes, as model biomaterial surfaces. The amount of serum Vn and Fn which adsorbed onto tissue culture grade polystyrene (TCP) from different serum concentrations over the range of 0.1-30% (v/v) were determined and compared to attachment of cells of the BHK-21 and HT1080 fibroblast lines. There was no simple correlation between the amount of Fn or the amount of Vn adsorbed and cell attachment and spreading. The requirement for Fn or Vn for attachment and spreading of BHK-21 or HT1080 cells onto modified polystyrene (either TCP or to Primaria) during the first 90 min of cell culture was directly tested by selective removal of Fn or Vn from the serum prior to addition to the culture medium. Attachment and spreading of BHK-21 or HT1080 cells onto TCP or Primaria surfaces were reduced in a concentration-dependent manner when the cells were seeded in medium containing 2% (v/v) or higher concentrations of Vn-depleted serum. BHK-21 cells or HT1080 cells seeded in medium containing Fn-depleted serum (which contained Vn) attached and spread onto TCP or Primaria. Both BHK-21 cells and HT1080 cells failed to attach to TCP or Primaria when seeded in medium containing serum depleted of both Vn and Fn. The requirement for serum Vn or Fn for fibroblast attachment to TCP was also tested using cells of a human dermal fibroblast strain. The attachment of the dermal fibroblasts to TCP during the first 90 min of culture was not decreased by depletion of Vn from the 15% (v/v) serum, but there was a reduction in the proportion of the attached cells which had spread. Selective depletion of serum Fn did not have any effect on either cell attachment or spreading. Our results show that for fibroblast cells, particularly with cell lines such as BHK-21 or HT1080 but also with cell strains, the first binding of cells onto tissue culture polystyrene when plated in medium containing serum is a result of adsorption onto the surface of serum Vn. The adsorption of serum Vn onto the surface overcomes the effect of serum components which tend to decrease cell attachment.


Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association. Wild-type and mutant chicken integrin beta 1 subunit (beta 1c) cDNAs were expressed in NIH 3T3 cells and assayed for localization in focal adhesions of cells plated on fibronectin substrates. Focal adhesion localization in stable transfected cells was assayed by indirect immunofluorescent staining with chicken-specific anti-beta 1c antibodies. Mutant beta 1c integrins containing internal deletions of 13 amino acids adjacent to the membrane, delta 759-771, and 20 centrally located amino acids, delta 771-790, localized in focal adhesions demonstrating that sequences required for direction to focal adhesion structures were not limited to one region of the cytoplasmic domain. Point mutations revealed three clusters of amino acids which contribute to localization in focal adhesions. These three clusters or signals are: cyto-1 (764-774), cyto-2 (785-788), and cyto-3 (797-800). The 11-residue cyto-1 signal is only found on integrin beta subunit sequences, except beta 4. Four residues within this region, D764, F768, F771, and E774, could not be altered without reducing focal adhesion staining intensities, and likely form a signal that occupies one side of an alpha helix. Mutations involving two cyto-1 residues, K770 and F771, also appeared to affect heterodimer affinity and specificity. Cyto-2 (785-788,), NPIY, is an NPXY signal that forms a tight turn motif. Cyto-2 provides a structural conformation, which when perturbed by proline removal or addition, inhibits integrin localization in focal adhesions. Cyto-3 (797-800), NPKY, resembles cyto-2, however, the nonconserved proline residue can be replaced without alteration of the localization phenotype. Cyto-3, therefore, constitutes a unique integrin signal, NXXY. Both serine and tyrosine residues at positions 790 and 788, respectively, which have been implicated in integrin phosphorylation/regulation, were conservatively replaced without detectable effect on focal adhesion localization. However, acidic replacements for these amino acids reduced focal adhesion staining intensities, suggesting that phosphorylation at these sites may negatively regulate integrin function.


Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility. A cDNA encoding a unique hyaluronan receptor has been molecularly cloned from a lambda GT11 3T3 cDNA expression library. Immunoblot analyses of cell lysates, using antibodies to peptides encoded in the cDNA, specifically react with a 58-kD protein. This protein is regulated by the mutant H-ras gene in cells containing a metallothionein promoter H-ras hybrid gene. Further, antibodies to peptide sequences encoded in the cDNA block the increase in locomotion resulting from induction of the mutant H-ras gene in this cell line. In a transblot assay, the bacterially expressed protein binds to biotinylated hyaluronan. Antibodies to peptides encoded in the cDNA react in immunoblot assays with the 58- and 52-kD proteins of a novel hyaluronan receptor complex previously implicated in cell locomotion. Furthermore, antibodies specific to the 58- and 52-kD proteins, which block ras-induced locomotion, also cross-react with the expressed, encoded protein. The gene product described here appears to be a new type of hyaluronan receptor that is involved in cell locomotion. It is named RHAMM, an acronym for receptor for hyaluronan-mediated motility.


Expression of fibronectin and integrins in cultured periodontal ligament epithelial cells. The process of attachment of epithelial cells obtained from the porcine periodontal ligament (cell rests of Malassez) to different extracellular matrix proteins and their expression of fibronectin and integrin receptors were studied by means of immunocytochemistry, in situ hybridization, and time-lapse cinemicrography techniques. The cell lines of periodontal ligament epithelial cells (PLE cells) attached to and spread rapidly on fibronectin, vitronectin, and type I collagen. One of the cell lines also attached to laminin, while the other cell line showed poor attachment to both laminin and Matrigel, a basement membrane material. By use of the in situ hybridization technique, some PLE cells were found to express the fibronectin gene strongly. Immunocytochemical staining localized fibronectin in extracellular fibrils and intracellular granules. Fibronectin was also found in the tracks left behind by the cells migrating on the substratum. Arg-gly-asp-ser peptide inhibited the attachment of the PLE cells to fibronectin, laminin, type I collagen, and vitronectin by 47%, 43%, 83%, and 94%, respectively, suggesting that the cell-matrix interactions were partly mediated by receptors related to the integrin family. Antibodies against the beta 1-integrin subunit stained the cell bodies and the plasma membrane projections of spreading cells. After 24 h or longer in culture, beta 1-integrins were localized to the regions of cell-cell contact. Cinemicrography of the arg-gly-asp-ser-peptide-treated cells demonstrated that the spreading and migration of isolated cells were prevented by the peptide. The peptide did not appear to dissociate the cell-cell contacts or interfere with migration of spread-cell colonies.(ABSTRACT TRUNCATED AT 250 WORDS)