Extracellular matrix vesicle distribution in primary mineralization two weeks after injury to rat tibial bone (ultrastructural tissue morphometry).

Primary mineralization on the 14th day of bone healing served as a model to study the distribution of extracellular matrix vesicles by means of transmission electron microscopy combined with computerized morphometry. Vesicles were traced on electron micrographs and classified according to diameter, distance from the calcified front, and type. The different types were determined as follows: electron-lucent vesicles ("empty"), vesicles with amorphous contents ("amorphic"), vesicles containing crystalline depositions ("crystal"), and vesicles with crystals and ruptured membranes ("rupture"). The majority of the vesicles measured between 0.02 and 0.07 micron and were located at a distance of less than 3 micron from the calcified front. They were distributed according to "empty", "amorphic", "crystal" and "rupture" type in concentrations of 10%, 31%, 51% and 8%, respectively. The diameters of the "rupture" vesicles were significantly larger than those of the "empty" and "amorphic" types. The sequence of their location, starting at the calcified front, ran as follows: "rupture", "crystal", "amorphic" and "empty", with the "rupture" type proximate to the front. According to the working hypothesis on calcification via extracellular matrix vesicles, it is thought that the "empty" vesicles are secreted by the cell and that subsequently amorphous Ca and Pi accumulate intravesicularly to form a hydroxyapatite crystal which, in turn, brings about rupture of the vesicle's membrane. The results of the present study support this theory and, additionally, show that the maturation process is accompanied by an increase of the vesicular diameter and by its approximation to the calcifying front.

Beneficial effects of corticosteroids on ocular myasthenia gravis.

OBJECTIVE: To determine if moderate-or low-dose corticosteroid therapy can reduce the diplopia and frequency of deterioration to generalized disease in ocular myasthenia gravis. DESIGN: Retrospective record review. SETTING: Two university-based neuro-ophthalmology services. PATIENTS: All 32 patients with ocular myasthenia gravis, treated with prednisone, followed up for a minimum of 2 years were included. Patients were treated with 1 or more courses of daily prednisone (highest initial dose, 40-80 mg) gradually withdrawn over 4 to 6 weeks. Subsequently, in 6 patients, 2.5 to 20 mg of prednisone was given on alternate day. OUTCOME MEASURES: Diplopia in the primary position or downgaze diplopia and generalized myasthenia gravis after 2 years of follow-up. RESULTS: Diplopia, which was initially found in the primary position in 29 patients and in the downgaze position in 26 patients, was absent in 21 patients at 2 years. Generalized myasthenia gravis occurred in 3 patients at 2 years. Elevated serum acetylcholine receptor antibody levels and abnormal electromyography findings were not predictive of worsening. No patient experienced a major steroid complication. CONCLUSIONS: Moderate-dose daily prednisone for 4 to 6 weeks, followed by low-dose alternate-day therapy as needed, can control the diplopia in patients with ocular myasthenia gravis. The frequency of deterioration to generalized myasthenia gravis at 2 years may be reduced; 9.4% in this study compared with more than 40% previously reported frequency. Corticosteroids may be useful even when ocular motor dysfunction is not normalized.

Neuroleptic malignant syndrome caused by dopamine-depleting drugs in a patient with Huntington disease.

Prior reports of neuroleptic malignant syndrome (NMS) concerned patients with psychiatric disorders, usually schizophrenia, who were taking dopamine receptor blocking agents. We report the syndrome in a patient with Huntington disease who was treated with dopamine-depleting agents. He had a negative evaluation for malignant hyperthermia (MH), and we suggest that NMS differs from MH. The occurrence of NMS caused by dopamine-depleting agents suggests that anticholinergic properties of phenotiazines are not the only cause. Central dopaminergic systems probably participate in thermoregulation, and dopamine depletion probably plays a pathogenetic role in this syndrome.

Changes in the distribution of extracellular matrix vesicles during healing of rat tibial bone (computerized morphometry and electron microscopy).

A study of the distribution of extracellular matrix vesicles during the first 3 weeks of healing of adult rat tibial bone was performed by transmission electron microscopy in combination with computerized morphometry. Bone injury comprised removal of the marrow followed by regeneration of the tissue via a phase of primary mineralization. A total number of 39,498 vesicles were traced on electron micrographs and sorted according to their diameters, distance from the calcified front and types. The different vesicular types were defined as follows: (a) vesicles with electron lucent contents, i.e. "empty", (b) vesicles with amorphous electron opaque contents, i.e. "amorphic", (c) vesicles containing crystalline depositions, i.e. "crystal", and (d) vesicles containing crystalline structures with ruptured membranes i.e. "rupture". The vesicles were studied on the days 3, 6, 14 and 21 after bone injury. Most of the vesicles were concentrated between diameters of 0.07 and 0.17 micron. Most of the vesicles were found in a distance less than 3 microns from the calcified front. The sequence of changes of distances from the calcified front and of the vesicular diameters were recorded as follows: "rupture", "crystal", "amorphic" and "empty", the "rupture" type being the closet to the front and of the largest diameter in each day. The results of the present study confirm the accepted hypothesis on calcification via extracellular matrix vesicles. It is thought that the cell secretes "empty" vesicles that accumulate Ca and Pi forming amorphous calcium phosphate that is then converted to hydroxyapatite. This is followed by rupture of the vesicular membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Very high mutation rate in offspring of Chernobyl accident liquidators.

Exposure to ionizing radiation has long been suspected to increase mutation load in humans. Nevertheless, such events as atomic bombing seem not to have yielded significant genetic defects. The Chernobyl accident created a different, long-term exposure to radiation. Clean-up teams (or 'liquidators') of the Chernobyl reactor are among those who received the highest doses, presumably in some combination of acute and chronic forms. In this study, children born to liquidator families (currently either in the Ukraine or Israel) conceived after (CA) parental exposure to radiation were screened for the appearance of new fragments using multi-site DNA fingerprinting. Their sibs conceived before (CB) exposure served as critical internal controls, in addition to external controls (non-exposed families). An unexpectedly high (sevenfold) increase in the number of new bands in CA individuals compared with the level seen in controls was recorded. A strong tendency for the number of new bands to decrease with elapsed time between exposure and offspring conception was established for the Ukrainian families. These results indicate that low doses of radiation can induce multiple changes in human germline DNA.

Molecular changes in the offspring of liquidators who emigrated to Israel from the Chernobyl disaster area.

The primary goal of this research was to reveal de novo mutations in the liquidators (cleanup personnel) who emigrated to Israel from the Chernobyl disaster area. We used genome fingerprinting simple sequence repeat-anchored polymerase chain reaction (PCR) amplification and random amplified polymorphic DNA PCR (RAPD PCR). The methodology involved a combination of RAPD PCR, polyacrylamide gel electrophoresis, and silver staining, with arbitrarily primed PCR. Use of microsatellite markers appears to be the most promising technique for high sensitivity analysis. The analysis involved DNA isolated from the blood of experimental and control subjects (involving both offspring who were born before or after the disaster and their parents). Our studies have reproducibly detected new bands that appeared in the children born after the disaster. No such bands appeared in the children born in the same family before the accident or in the children of control families who had not been exposed to radiation.

The role of hypovolemic stress in the production of fat embolism in rabbits. 2. Changes in arterial blood gas levels and static compliance.

The purpose of this study was to assess the possible role of hypovolemia as a factor in the production of impaired pulmonary function in pulmonary fat embolizaiton. Iv vivo static lung compliance was measured by inflating the lungs with known volumes of air and recording the transpulmonary pressure, monitoring intraesophageal pressure as an index of intrapleural pressure. Arterial blood was drawn from the central artery of the ear. Embolization was produced by the intravenous injection of homologous fat cells either as the sole experimental procedure or in conjunction with hypovolemic shock. Two types of shock were studied. In one, 20 percent of the blood volume was removed, producing hypovolemia associated with hemodilution, and in the other a tourniquet was applied to induce hypovolemia and hemoconcentration. The results indicate that embolization is associated with significantly greater impairment of pulmonary function if it develops on a background of shock than if it occurs in an otherwise healthy animal. These pathophysiologic findings parallel the results of a morphologic study.

The role of hypovolemic stress in the production of fat embolism in rabbits. 1. Morphologic alterations of the lungs.

In rabbits, an experimental model of fat embolism was produced that simulates the course of events in the clinical situation. Small doses of fat-cell suspension (0.075 ml/kg of body weight), prepared by collagenase treatment of homologous adipose tissue, were injected intravenously. Concomitantly, hypovolemia was produced in two animal groups by either withdrawing 20 percent of the estimated blood volume or by application of a hind-limb ischemic tourniquet for 90 minutes. The presence of pathoanatomic characteristics typical of fat embolism was evaluated by recording lung/body weights, macroscopic appearance, and semiquantitative microscopic estimation in the lungs of edema, hemorrhage, atelectasis, intravascular coagulation, and leukocytic thrombi. Mean indices of lung/body weight were higher in all animals receiving injections of fat-cell suspension, as compared to controls. The score for microscopic generalized pulmonary damage was significantly higher in rabbits exposed to both fat-cell injections and hypovolemia than in controls or after fat-cell injections alone. It is concluded that hypovolemia enhances the development of fat embolism in rabbits subjected to small doses of fat-cell suspension.

The use of a squid third order spatial gradiometer to measure magnetic fields of the brain.

It appears to be clear from the results that the third order gradiometer is able to detect small biomagnetic signals from the brain which are related to evoked potentials and spontaneous electrical activity. The instrument operates reasonably well within a noisy environment, however further development is necessary to balance the first gradient. We intend to pursue this direction with software systems. Some of the data presented suggest that components of MEG evoked activity may change independently of EEG. One interpretation which may derive from this is that the same current dipoles are probably not responsible for the entire configuration of evoked fields. This interpretation is consistent with EEG evidence which indicates that analogous components in the evoked potential may vary independently as a function of stimulus parameters and information processing. Perhaps a model of magnetic dipoles due to small current loops would be more compatible with the electrophysiological data.

Neural network classifications and correlation analysis of EEG and MEG activity accompanying spontaneous reversals of the Necker cube.

It has recently been suggested that reentrant connections are essential in systems that process complex information [A. Damasio, H. Damasio, Cortical systems for the retrieval of concrete knowledge: the convergence zone framework, in: C. Koch, J.L. Davis (Eds.), Large Scale Neuronal Theories of the Brain, The MIT Press, Cambridge, 1995, pp. 61-74; G. Edelman, The Remembered Present, Basic Books, New York, 1989; M.I. Posner, M. Rothbart, Constructing neuronal theories of mind, in: C. Koch, J.L. Davis (Eds.), Large Scale Neuronal Theories of the Brain, The MIT Press, Cambridge, 1995, pp. 183-199; C. von der Malsburg, W. Schneider, A neuronal cocktail party processor, Biol. Cybem., 54 (1986) 29-40]. Reentry is not feedback, but parallel signalling in the time domain between spatially distributed maps, similar to a process of correlation between distributed systems. Accordingly, it was expected that during spontaneous reversals of the Necker cube, complex patterns of correlations between distributed systems would be present in the cortex. The present study included EEG (n=4) and MEG recordings (n=5). Two experimental questions were posed: (1) Can distributed cortical patterns present during perceptual reversals be classified differently using a generalised regression neural network (GRNN) compared to processing of a two-dimensional figure? (2) Does correlated cortical activity increase significantly during perception of a Necker cube reversal? One-second duration single trials of EEG and MEG data were analysed using the GRNN. Electrode/sensor pairings based on cortico-cortical connections were selected to assess correlated activity in each condition. The GRNN significantly classified single trials recorded during Necker cube reversals as different from single trials recorded during perception of a two-dimensional figure for both EEG and MEG. In addition, correlated cortical activity increased significantly in the Necker cube reversal condition for EEG and MEG compared to the perception of a non-reversing stimulus. Coherent MEG activity observed over occipital, parietal and temporal regions is believed to represent neural systems related to the perception of Necker cube reversals.

Testing for neurogenic impotence: a challenge.

The bulbocavernosus reflex and pudendal evoked responses were obtained in a group of control patients and impotent men in an attempt to assess their reliability and objectivity in diagnosing neurogenic erectile dysfunction.

Carcinomatous meningitis from transitional cell carcinoma of bladder.

The following case report presents a patient with transitional cell carcinoma of the bladder referred to this medical center after carcinomatous meningitis developed. Previously he had undergone surgical resection of the primary lesion and had received cis-platinum chemotherapy for lung metastasis. This unusual presentation of metastatic disease (carcinomatous meningitis) seems to alert the surgical and medical communities to new complications.

Studies on the control of myelinogenesis. II. Evidence for neuronal regulation of myelin production.

Tritiated thymidine has been used as a nuclear marker to trace the origin of Schwann cells, sited in the distal stump of a severed unmyelinated nerve, which are able to elaborate myelin around axons regenerating from an anastomosed proximal stump of a severed myelinated nerve. Two types of cross-anastomosis experiments were performed in young, adult rats: (1) the proximal stump of a myelinated sternohyoid nerve was labeled (5 mCi/kg body weight) selectively over a 4-day period of predetermined maximal thymidine uptake and two days later, after flushing the animal repeatedly with cold thymidine, the unmyelinated cervical sympathetic trunk was transected and its unlabeled distal stump linked to the proximal stump of the labeled sternohyoid nerve; (2) the distal stump of an unmyelinated cervical sympathetic trunk was labeled selectively over a 5-day period of predetermined maximal uptake and two days later, after flushing with cold thymidine, the myelinated sternohyoid nerve was severed and its unlabeled proximal stump linked to the labeled distal stump of the cervical sympathetic trunk. The fate of the labeled cells in each type of anastomosis was determined 3 weeks later by autoradiography and liquid scintillation spectrometry. In the first type, a small amount of label had migrated from proximal stumps but labeled Schwann cells were not found in successfully anastomosed distal stumps. In the second type, labeled Schwann cells were seen in the cervical sympathetic trunk in association with myelinated and non-myelinated axons regenerating from the sternohyoid nerve. These data suggest that the presence or absence of myelin formation by a Schwann cell is controlled by some property of the axon with which it is associated. Putative mechanisms underlying neuronal control of myelinogenesis are discussed.

Sensory feedback contributes to early movement-evoked fields during voluntary finger movements in humans.

Neuromagnetic field changes accompanying voluntary movement in humans ('movement-evoked fields' or MEFs) were recorded over the scalp using a whole-head MEG system during the performance of self-paced finger movements in order to determine the contribution of sensory feedback to the generation of these brain responses. It was found that cooling the subject's arm resulted in delays of 8 ms or more in the latency of the early movement-evoked field component (MEFI). These delays were attributed to increases in conduction times in the afferent pathways as confirmed by electrically evoked somatosensory responses and suggest a peripheral origin of the MEFI. In a second experiment, we demonstrated the effects of sensory input to the contralateral hand during a simple button pressing task in 4 subjects. The results indicated that responses over the hemisphere ipsilateral to the side of movement which resembled previously reported ipsilateral MEFs can be elicited by the spread of mechanical stimulation to opposite side of the body when a mechanical trigger is used. These experiments provide further evidence that early movement-evoked fields produced by unilateral finger movements are observed primarily over the contralateral somatosensory cortex and represent sensory feedback to the somatosensory cortex from the periphery.

Anin vivo method to prepare normal Schwann cells free of axons and myelin.

A viable population of undifferentiated Schwann cells may be prepared from chronically denervated peripheral nerves. Nerve transection stimulates a sequence of cellular events in distal stumps leading to removal of axons and myelin, and proliferation of Schwann cells. Sealing the ends of nerve stumps prevents reinnervation and leaves daughter Schwann cells residing in longitudinal columns. Schwann cells may be harvested from the endoneurial tissue of the nerve stumps 5-12 weeks after nerve transection. Unlike myelinating cells prepared from intact tissue, where function has been specified by associated axons, Schwann cells obtained from denervated stumps are functionally naive. Their usefulness in analyzing axonal regulation of myelinogenesis and mitosis is therefore suggested.

Motor cortex activity and predicting side of movement: neural network and dipole analysis of pre-movement magnetic fields.

Neuromagnetic fields were recorded from human subjects during the performance of left and right voluntary finger movements. Modeling of current dipole sources indicated symmetric activation of both motor cortices beginning 600 ms prior to movement onset. This activity became lateralized to the contralateral hemisphere 200-300 ms prior to movement onset, the period during which an artificial neural network showed increased ability to predict side of movement within single trials. The results describe the mechanism of lateralization of cortical brain activity preceding voluntary movement and provide further evidence of the involvement of ipsilateral motor cortex in unilateral movements.

New, sensitive and selective method for determining sub-microgram/l levels of bromate in drinking water.

Health effects studies suggest that bromate should be regulated at 0.5 microgram/l or less in drinking water. Accordingly, an analytical method is needed to quantify this contaminant with great sensitivity and selectivity. Past efforts to do this have focused on pre-concentration ion chromatographic (IC) separation followed by suppressed conductivity detection. Interfering chloride was removed by passing samples over a silver resin which increased sample analysis time to almost 1 h. In this paper, a new method which uses IC separation with no pre-treatment followed by a post-column reaction to produce tribromide (Br3-) from bromate, is applied to the analysis of a variety of aqueous samples. The tribromide ion is detected by UV absorbance at 267 nm. This method is very sensitive for bromate with a limit of quantitation of 0.2 microgram/l and is also very selective. Common anions typically separated by IC exhibit no interference, even at the levels normally found in drinking water.

Use of ion chromatography with post-column reaction for the measurement of tribromide to evaluate bromate levels in drinking water.

A user-friendly ion chromatography method in conjunction with a post-column reaction (PCR) achieves practical quantitation limits for the oxyhalides bromate and chlorite of 0.05 microg/l and 0.10 microg/l, respectively. This level of measurement allows for the accurate assessment of bromate contributed to finished drinking waters that have been chlorinated using sodium hypochlorite. The target sensitivity of oxyhalides in the presence of other major ion species typically found in drinking water is achieved by PCR using excess bromide under acidic conditions to form a tribromide species that is detected by ultraviolet spectrometry. The method setup involves non-hazardous materials, as opposed to other recently developed methods that employ somewhat hazardous chemicals for generating the reaction necessary for the detection of bromate at sub-microg/l levels. No pretreatment of the samples is required, other than filtration and quenching of oxidant residual.

Brain potentials, perceptual mechanisms and semantic categorisation.

The behaviour of intermediate and long-latency evoked potential (EP) components were investigated in a semantic categorisation task. Visually presented 'narrow' and 'wide' priming category questions, intended to vary processing load, were followed, after 1.5 sec by a 100 msec presentation of a positive or negative instance for decision. Neither behavioural response latencies nor amplitudes of N1-P2 (130 and 212 msec mean latencies) differentiated between 'narrow' and 'wide' category decisions. However, response latencies were faster and N1-P2 amplitudes greater for positive than for negative instances of primed categories. This result is attributed to the priming effect, by 'spreading activation, of prior category questions on 'logogen-like' pre-conscious word detecting devices within the brain. Semantic discrimination by N1-P2 contradicts previous assertions that intermediate components only reflect selection based on crude stimulus characteristics. Comparison of N1-P2 amplitudes at lateral electrodes offered no evidence of hemispheric lateralisation of language function in the experimental task. There was no late wave unequivocally identifiable as P300 on morphological and scalp distribution criteria. A positive peak at mean latency 327 msec (called P3) was of low amplitude and stable latency and did not appear to index processing load as defined by the experimenters. However, the amplitude of N2-P3 was larger to positive instances of narrow categories than any of the other three conditions. This could be the sort of probability effect seen in P300 behaviour if it is assumed that the brain can discriminate between stimuli on the basis of the size of the subset from which they come.

The contingent negative variation (CNV) and speech production: slow potentials and the area of Broca.

The lateral distribution of the CNV was investigated during conditions that required normal male and female subjects to vocalize simple English words in a CNV paradigm. In addition to a non-verbal or comparison condition (Tone-Clicks/Button Press), two word conditions presented stimulus words in either S1 or S2 positions (Word-Tone/Speak; Tone-Word/Speak) and a third condition required a one-word association to the stimulus words (Word-Tone/Speak Association). Monopolar scalp activity was collected from sites that included a location approximating Broca's speech area on the left hemisphere, a homologous comparison site on the right hemisphere, frontal sites (F3, F4) and the vertex (Cz). Although several subjects showed some asymmetrical activity in some of the word conditions, amplitude measures based on CNV averages indicated no consistent lateralization effects over the left hemisphere prior to word vocalizaiton. Negative pretrial shifting was suspected in those conditions in which words appeared as S1 signals.