Clinical correlates of dental anxiety among older adults.

Most studies of the relationship between dental anxiety and oral health status have used subjective rather than clinical indicators of oral health. In this paper we examine differences between older adults who are and are not dentally anxious using a broad range of clinical measures. The data show that dentally anxious individuals were more likely to be edentulous, and among the dentate, had more missing and fewer filled teeth. As a result, dentally anxious dentate subjects were more likely to need prosthodontic treatment. They were also more likely to need immediate treatment for the relief of pain and infection and periodontal care. There was evidence to suggest differences in patterns of dental treatment between those who were and were not dentally anxious and some evidence consistent with the hypothesis that aging influences the relationship between dental anxiety and oral health status.


Oral self-care behaviours in older dentate adults.

Defining oral self-care as activities undertaken by individuals to maintain or promote dental health, this paper examines the distribution of oral self-care behaviours in a sample of dentate adults, age 50 and over, living in Ontario, Canada (n = 713). Results are presented separately for two older cohorts, respondents ages 50-64 and those 65 and older, and for females and males. Six oral self-care behaviours were examined: toothbrushing, flossing, use of an interdental device, extra fluoride use, frequency of between meal snacks and ingestion of cariogenic foods. High rates of self-care occurred only for toothbrushing. Age groups differed significantly for use of interdental devices, consumption of cariogenic foods and snacking between meals while gender differences were found for toothbrushing, flossing and consuming sweet foods. The need to target older adults and especially older males for oral hygiene information and instruction in order to counteract possible misleading beliefs learned before the advent of current dental hygiene practices is apparent from these data.


Diastolic heart failure.

Diastolic heart failure is a distinct clinical entity increasingly seen in older patients and requires special awareness to make the diagnosis. Although no single laboratory test is identified for making a confident diagnosis of diastolic dysfunction as the pathogenetic mechanism for heart failure, a constellation of echocardiographic and radionuclear findings are helpful in most cases. Invasive assessment of LV diastole is laborious, requiring high-fidelity pressures and accurate measures of volumes, and these are rarely needed to diagnose the condition. It appears that prognosis is significantly better for those with normal systolic function, when compared with congestive heart failure caused by impaired systolic pump function. Finally, the therapeutic approaches are substantially different for the two groups. It must be emphasized that even patients with predominant myocardial systolic dysfunction have some combined diastolic dysfunction as well. This latter group is difficult to treat. However, improvement in systolic pump function, when markedly impaired, must take precedence in management strategies.


Glycation products and the pathogenesis of diabetic complications.

Glucose irreversibly modifies long-lived macromolecules by forming AGEs as a function of glucose concentration and time. AGEs cause qualitative and quantitative changes in extracellular matrix components such as type IV collagen, laminin, and vitronectin. These AGE-induced changes can affect cell adhesion, growth, and matrix accumulation. AGE-modified proteins also alter cell function by interacting with specific receptors on macrophages and endothelial cells, inducing changes that promote matrix overproduction, focal thrombosis, and vasoconstriction. DNA and nuclear proteins also may be targets for AGE damage. The persistence of accumulated AGEs during periods of normal glucose homeostasis may explain the phenomenon of hyperglycemic memory. Pharmacological inhibition of in vivo AGE formation by aminoguanidine prevents or ameliorates diabetic retinopathy, nephropathy, and neuropathy in animal models. These data suggest that aminoguanidine and other AGE inhibitors have a potential therapeutic role in the treatment of diabetic patients.


Diabetic retinopathy. A clinical overview.

After an introductory summary, this review presents a description of the natural course of diabetic retinopathy, then briefly considers selected pathogenetic and epidemiological concepts, and concludes with a discussion of current treatment.


Epidemiology of proliferative diabetic retinopathy.

This review examines recent epidemiological data about the prevalence and incidence of and risk factors for proliferative diabetic retinopathy. In addition, the relation of proliferative retinopathy to other systemic complications associated with diabetes is reviewed. The data come mostly from the baseline and 4-yr follow-up examinations of a large population-based study, the WESDR, which involved 996 younger-onset insulin-dependent people whose diabetes was diagnosed at < 30 yr of age and 1370 older-onset people whose diabetes was diagnosed at > or = 30 yr of age, and who were taking or not taking insulin. The major finding is that proliferative retinopathy is a prevalent complication (23% in the WESDR younger-onset group, 10% in the WESDR older-onset group that takes insulin, and 3% in the group that does not take insulin). Hyperglycemia, longer duration of diabetes, and more severe retinopathy at baseline were associated with an increased 4-yr risk of developing proliferative retinopathy. However, higher blood pressure at baseline was associated only with the development of proliferative retinopathy in the younger-onset group. The presence of proliferative diabetic retinopathy was associated with an increased 4-yr risk of loss of vision, cardiovascular disease, diabetic nephropathy, and mortality. In the WESDR, a significant number of diabetic people with proliferative retinopathy at risk for vision loss were not under the care of an ophthalmologist or had not undergone panretinal photocoagulation. These data suggest that hyperglycemia and, possibly, high blood pressure are related to proliferative retinopathy. They also suggest that once proliferative diabetic retinopathy is detected, people should have a medical evaluation, because it is a strong indicator for the presence and development of systemic disease. These data also indicate that diabetic patients and their physicians should be aware of the need for routine ophthalmological examinations to detect and treat proliferative retinopathy.


Current hypotheses for the biochemical basis of diabetic retinopathy.

Diabetic retinopathy is one of the leading causes of vision loss in industrialized countries. Despite recent advances, the biochemical basis for the development of this diabetic complication is uncertain. Although retinal circulation is unique in that it is readily observable noninvasively, retinal tissue is extremely difficult to study in humans because of the problems inherent in obtaining fresh, appropriate biopsy material. Moreover, because of the difficulties in working with animal models of diabetic retinopathy, such as the dog, many investigators have turned to cell-culture models, especially those using primary cultures of retinal capillary endothelial cells and pericytes. Diabetic retinopathy involves both morphological and functional changes in the retinal capillaries. Morphological changes include basement membrane thickening and pericyte disappearance; functional changes include one important early change--increased permeability--which may be attributable to endothelial cell changes and basement membrane leakiness. Investigators have described major biochemical changes in cellular signaling pathways, including myo-inositol, inositol phosphates, and DAG metabolism, as well as decreased Na(+)-K(+)-ATPase and increased PKC activity. These defects may be related to the way endothelial cells and pericytes synthesize and interact with the extracellular matrix. Abnormalities in endothelial cell or pericyte interaction with the basement membrane may in turn lead to functional abnormalities, such as increased permeability.


Complications: neuropathy, pathogenetic considerations.

The most common form of neuropathy associated with diabetes mellitus is distal symmetric sensorimotor polyneuropathy, often accompanied by autonomic neuropathy. This disorder is characterized by striking atrophy and loss of myelinated and unmyelinated fibers accompanied by Wallerian degeneration, segmental, and paranodal demyelination and blunted nerve fiber regeneration. In both humans and laboratory animals, this progressive nerve fiber damage and loss parallels the degree and/or duration of hyperglycemia. Several metabolic mechanisms have been proposed to explain the relationship between the extent and severity of hyperglycemia and the development of diabetic neuropathy. One mechanism, activation of the polyol pathway by glucose via AR, is a prominent metabolic feature of diabetic rat peripheral nerve, where it promotes sorbitol and fructose accumulation, myo-inositol depletion, and slowing of nerve conduction by alteration of neural Na(+)-K(+)-ATPase activity or perturbation of normal physiological osmoregulatory mechanisms. ARIs, which normalize nerve myo-inositol and nerve conduction slowing, are currently the focus of clinical trials. Other specific metabolic abnormalities that may play a role in the pathogenesis of diabetic neuropathy include abnormal lipid or amino acid metabolism, superoxide radical formation, protein glycation, or potential blunting of normal neurotrophic responses. Metabolic dysfunction in diabetic nerve is accompanied by vascular insufficiency and nerve hypoxia that may contribute to nerve fiber loss and damage. Although major questions about the pathogenesis of diabetic neuropathy remain unanswered and require further intense investigation, significant recent progress is pushing us into the future and likely constitutes only the first of many therapies directed against one or more elements of the complex pathogenetic process responsible for diabetic neuropathy.


Diabetic neuropathies.

Diabetic neuropathy is a common complication of diabetes that may be associated both with considerable morbidity (painful polyneuropathy, neuropathic ulceration) and mortality (autonomic neuropathy). The epidemiology and natural history of diabetic neuropathy is clouded with uncertainty, largely caused by confusion in the definition and measurement of this disorder. We have reviewed various clinical manifestations associated with somatic and autonomic neuropathy, and we herein discuss current views related to the management of the various abnormalities. Although unproven, the best evidence suggests that near-normal control of blood glucose in the early years after diabetes onset may help delay the development of clinically significant nerve impairment. Intensive therapy to achieve normalization of blood glucose also may lead to reversibility of early diabetic neuropathy, but again, this is unproven. Our ability to manage successfully the many different manifestations of diabetic neuropathy depends ultimately on our success in uncovering the pathogenic processes underlying this disorder. The recent resurgence of interest in the vascular hypothesis, for example, has opened up new avenues of investigation for therapeutic intervention. Paralleling our increased understanding of the pathogenesis of diabetic neuropathy, refinements must be made in our ability to measure quantitatively the different types of defects that occur in this disorder. These tests must be validated and standardized to allow comparability between studies and more meaningful interpretation of study results.


Electrophysiologic studies of cervical vagus nerve stimulation in humans: II. Evoked potentials.

Evidence from studies of experimental animals indicates that electrical stimulation of the vagus nerve not only can alter the EEG but evokes activity in specific brain areas. We report effects of electrical stimulation of the vagus nerve in 9 patients with medically intractable seizures as part of a clinical trial of chronic vagal stimulation for control of epilepsy. The left vagus nerve in the neck was stimulated with a programmable implanted stimulator. Effects of stimulus amplitude, duration, and rate were studied. Noncephalic reference recording of the vagus nerve evoked potential showed some unusual properties: a scalp negative component occurred with a latency of 12 ms, very high amplitude (< or = 60 microV), and widespread scalp distribution. Field distribution studies indicated that this potential was myogenic in origin and generated in the region of the stimulating electrodes in the neck area. Chemically induced muscle paralysis confirmed this observation. Bipolar scalp recording showed several small-amplitude topographically distinct potentials occurring in 30 ms. No effect, either acute or chronic, could be detected on pattern-reversal evoked potentials, auditory brainstem evoked potentials, auditory 40-Hz potentials, or cognitive evoked potentials.


Seizures in a fetal lamb after cocaine exposure: a case report.

We report the chance observation of two generalized seizures in a fetal lamb that received cocaine. Cocaine was infused intravenously into a 138-day-old fetus while electrocortical (ECoG), electroocular (EOG), and neck electromyographic (EMG) activity were recorded. Fetal rapid eye movement (REM) and nonrapid eye movement (NREM) sleep were disrupted during the cocaine exposure and returned to a normal pattern after infusions were stopped. Seventy-two hours later, however, we recorded generalized seizures with ECoG spike and spike wave activity.


Electrophysiological studies of cervical vagus nerve stimulation in humans: I. EEG effects.

Evidence from studies of experimental animals indicates that electrical stimulation of the vagus nerve alters EEGs under certain stimulus parameters. We report EEG effects of electrical stimulation of the vagus nerve in 9 patients with medically intractable seizures as part of a clinical trial of chronic vagal stimulation for control of epilepsy. The mechanism of action of the vagal antiepileptic effect is unknown, and we believed that analysis of electrophysiologic effects of vagal nerve stimulation would help elucidate the brain areas affected. The left vagus nerve in the neck was stimulated with a programmable implanted stimulator. Stimulation at various stimulus frequencies and amplitudes had no noticeable effect on EEG activity whether the patient was under general anesthesia, awake, or asleep, but vagus nerve stimulation may interrupt ongoing ictal EEG activity.


Inhibition of experimental seizures in canines by repetitive vagal stimulation.

Repetitive electrical stimulation of the canine cervical vagus nerve interrupts or abolishes motor seizures induced by strychnine and tremors induced by pentylenetetrazol (PTZ). Tremors were defined as rhythmic alternating contractions of opposing muscle groups, exerting much less force than seizure contractions. Seizures were induced by injection boluses of strychnine or PTZ at 1- to 4-min intervals until sustained muscle activity was observed electromyographically (EMG). Vagal stimulation terminated seizures in 0.5-5 s. There were prolonged periods with no spontaneous EMG activity after stimulation. The period of protection was approximately four times the stimulation period. The antiseizure actions of vagal stimulation were not altered by transection of the vagus distal to the stimulating electrode. Optimal stimulus parameters were estimated: strength, approximately 20 V (electrode resistance 1-5 omega); frequency 20-30 Hz; duration, approximately 0.2 ms. These data suggest that the antiseizure effects derive from stimulation of small-diameter afferent unmyelinated fibers in the vagus nerve. These results may form the basis of a new therapeutic approach to epilepsy.


Pre- and postoperative auditory event-related potentials in temporal lobe epilepsy.

We studied auditory event-related potentials (ERPs) in 11 surgically treated patients with intractable temporal lobe epilepsy (TLE) pre- and postoperatively. ERPs through sphenoidal electrodes (Sp1-T3, Sp2-T4) provided clinically relevant and correctly lateralizing electrophysiologic evidence of temporal lobe dysfunction in 9 of 11 patients, confirmed by EEGs, electrocorticograms, neuroradiologic, and neuropsychological results, and clinical follow-up. Lateralizing asymmetries were noted in P300 amplitude, but latencies were prolonged bilaterally. Sphenoidal ERPs might serve as a new functional indicator of temporal lobe dysfunction in patients evaluated for epilepsy surgery.


Factors in children that predict performance on the intracarotid amobarbital procedure.

The intracarotid amobarbital procedure (IAP) is frequently used to determine hemispheric language dominance and memory competence in individuals with intractable epilepsy before surgical intervention. The present study focused on outcome results concerning use of this technique in children. The IAP was an effective tool in determining their hemispheric language dominance. Seven characteristics, including age, gender, dose level of sodium amobarbital, Full-Scale I.Q., hemispheric side of injection (left or right), language dominance of hemisphere injected, and order of injection (first or second) were examined as possible factors predictive of memory performance on the IAP. Results suggest that after injection of the hemisphere suspected of containing the primary seizure origin, children aged < 13 years who had the left language-dominant hemisphere injected were significantly less likely to pass IAP memory tasks than were older children or younger children who had the nondominant hemisphere injected. These findings suggest that the IAP is a reliable indicator of hemispheric memory competence in children aged > 13 years or younger children whose suspected seizure focus is located in the nondominant language hemisphere.


Prolonged ictal amnesia with transient focal abnormalities on magnetic resonance imaging.

A previously healthy woman had a prolonged amnestic state caused by complex partial status epilepticus with bilateral mesiotemporal lobe involvement confirmed by EEG with nasopharyngeal electrodes. A magnetic resonance imaging (MRI) scan obtained shortly after recovery from the amnesia showed reversible focal abnormalities consisting of increased signal intensity on T2-weighted scan in the mesiotemporal lobe.


Adult postanoxic "erratic" status epilepticus.

A 66-year-old woman with posttraumatic anoxic coma after diffuse cerebral fat embolism had continuous alternating-side myoclonic jerks. Usually, this kind of myoclonic status epilepticus (SE) occurs in newborn infants. We postulate the unusual combination of diffuse cerebral anoxia plus commissural fiber damage as a possible explanation.


Prevalence and incidence of epilepsy in Ulanga, a rural Tanzanian district: a community-based study.

A random cluster sample survey of approximately 18,000 people in 11 villages was performed in Ulanga, a Tanzanian district with a population of approximately 139,000 people. Well-instructed fourth-year medical students and neurologic and psychiatry nurses identified persons with epilepsy using a screening questionnaire and sent them to a neurologist for detailed evaluation. Identified were 207 subjects (88 male, 119 female) with epilepsy; of these, 185 (89.4%) (80 male, 105 female) had active epilepsy. The prevalence of active epilepsy was 10.2 in 1,000. Prevalence among villages varied, ranging from 5.1 to 37.1 in 1,000 (age-adjusted 5.8-37.0). In a 10-year period (1979-1988) 122 subjects living in the 11 villages developed epilepsy, with an annual incidence of 73.3 in 100,000. Generalized tonic-clonic seizures (GTCS) accounted for 58% and partial seizures accounted for 31.9%, whereas in 10.1% seizures were unclassifiable. Of the partial seizures, secondarily generalized seizures were the most common. Possible etiologic or associated factors were identifiable in only 25.3% of cases. Febrile convulsions were associated in 13.4 of cases. Other associated factors included unspecified encephalitis (4.7%), cerebral malaria (1.9%), birth injury (1.4%), and other (3%). In 38% of the cases, there was a positive family history of epilepsy.


Validation of a questionnaire for clinical seizure diagnosis.

A detailed questionnaire has been devised for diagnosis of seizure type. It is suitable for administration by trained interviewers, either directly or by telephone. A comparison of physician-based and questionnaire-based diagnoses showed almost perfect agreement in classification of patients into those with seizures of either generalized or focal origin. Substantial to almost-perfect agreement was reached in diagnosis of patients with most individual seizure types. Disagreement in differentiation between simple and complex partial seizures (CPS) probably reflects the limitations of the clinical method rather than of the questionnaire itself. The questionnaire should be of value in large-scaled clinical and epidemiologic studies.


Experience with the International League Against Epilepsy classifications of epileptic seizures (1981) and epilepsies and epileptic syndrome (1989) in epileptic children in a developing country.

Four hundred eighty-three epileptic children attending the Pediatric Epilepsy Clinic at Bai Jerbai Wadia Hospital for Children, Bombay, India were classified according to the International League Against Epilepsy (ILAE) classification of epileptic seizures (1981) and epilepsies and epileptic syndromes (1989). The predominant seizures were partial (53.6), generalized (40.3%), and unclassifiable (6%). In epilepsies and epileptic syndromes, 55.3% were partial, 27% were generalized, 13.5% were undetermined, and 4.1% were special syndromes. Although our results were similar in many respects to those of other reported series, some differences were observed in the incidence of partial and generalized seizures, and partial and generalized epileptic syndromes and their subgroups, such as idiopathic, symptomatic, and cryptogenic partial syndromes, idiopathic generalized syndromes, and symptomatic specific syndromes. These differences are probably due to different age limits, methods of case ascertainment and inclusion criteria, different genetic and environmental factors, variable interpretation of clinical and EEG features, and lack of facilities for investigation in developing countries. Despite various limitations, we were able to classify most cases; the ILAE classification can be used in developing countries so that comparison can be made with other studies.


Epilepsy and Persian culture: an overview.

This article reviews the manner in which Persian culture viewed the problem of epilepsy. Beginning with the Avesta, the earliest Persian text on health and sickness, the medical literature on treatments of epilepsy common in Iran are reviewed. The article also explores popular Persian concepts that try to explain the causes of the morbus sacer.


Epilepsy in Angelman syndrome associated with chromosome 15q deletion.

We report eight sporadic cases of typical Angelman syndrome (AS) associated with chromosome 15q12 deletion. Age at first visit was 3-35 months (average 18 months), and follow-up period was 4-20 years (average 14.1 years). The characteristic features of epilepsy in AS are (a) seizure onset in early childhood (8 of 8); (b) evolution of seizure type with age (8 of 8); (c) EEG abnormality changes from high-voltage slow bursts (HVS) in infancy to diffuse spike and waves in middle childhood (4 of 5); (d) atypical absence seizures (8 of 8), often occurring as atypical absence status (4 of 8); and (e) diminution of seizure discharges and clinical seizures after puberty (7 of 7). We believe that AS may frequently exist in the intractable epilepsies of childhood with severe mental retardation. We stress the importance of AS as one of the main etiologic background diseases of the intractable epilepsies with infantile onset such as West syndrome, Lennox-Gastaut syndrome, and others.


Angelman syndrome in three siblings: characteristic epileptic seizures and EEG abnormalities.

Neurologic findings in 3 siblings with Angelman syndrome (AS) with apparently normal karyotype but DNA deletion of 15q11-q12 deriving from their mother are described. Increased auditory brainstem response (ABR) thresholds were noted in all 3. Interictal EEG findings included periodic 2- to 3-Hz high-voltage slow wave bursts bioccipitally and sporadic slow spike wave complexes mainly bifrontally. EEG findings suggestive of minor epileptic status were apparent in the elder brother and may be a characteristic feature in young AS patients. Seizures suggestive of generalized epilepsy have been reported in 90% of AS patients. AS is considered a good model of symptomatic generalized epilepsy associated with chromosomal DNA deletion of the (GABA)A receptor beta 3-subunit gene.


Facial asymmetry in partial epilepsies.

Fifty-six consecutive epileptic patients with partial seizures (30 temporal, 26 extratemporal) and facial asymmetry were studied. Facial asymmetry was compared with EEG, radiologic, and other clinical findings. Thirty patients had a lesional epilepsy whereas 26 were considered cryptogenic. In lesional epilepsies, 60% of patients had EEG foci ipsilateral to the smaller hemiface and only 20% had EEG foci contralaterally. In the cryptogenic group, the EEG focus was ipsilateral in 50% and contralateral to the facial smallness in 46%. No differences were noted between temporal and extratemporal epilepsies. Early acquired cerebral lesions may modify development of the hemisphere involved, leading to a small ipsilateral hemiface and seizures originating from the same side.


Generalized spike-waves, multiple loci, and clinical course in children with EEG features of benign epilepsy of childhood with centrotemporal spikes.

In 41 patients with EEG features of benign epilepsy of childhood with centrotemporal spikes (BECCT), we noted associated generalized spike-wave discharges (GSWD) in 14.6% and multiple independent sharp wave foci in 9.8%. The presence or absence of these EEG features was not predictive of the clinical course. The high incidence of GSWD in children with BECCT suggests a possible relation in the expression of these two EEG traits.


Determinants of IQ profile in children with idiopathic generalized epilepsy.

To determine the IQ profile of children with epilepsy and the influence of various epilepsy-related variables on IQ scores, we studied 50 children with idiopathic generalized epilepsy of > 1-year duration, 25 of their siblings, and 30 healthy controls. IQ assessments were made with Malin's Indian modification of the Wechsler Intelligence Scale for Children. The mean +/- SD IQ scores of children with epilepsy (85.6 +/- 12) and their siblings (93.2 +/- 11) were significantly lower than those of the controls (101.6 +/- 9). The IQ scores of the children with epilepsy were also significantly lower than those of their siblings (p < 0.05). The IQ scores showed a significant correlation with socioeconomic status (SES) score (r = 0.33), a history of status epilepticus (r = -0.38), duration of seizure disorder (r = -0.31), and total number of seizures (r = -0.31). On multiple regression analysis, status epilepticus emerged as the most significant variable, accounting for 14% variance, followed by SES score (9% variance), duration of seizure disorder (6% variance), and sex of the child (5% variance). Genetic or environmental factors that probably lead to cognitive deficit in children with epilepsy and their siblings require further study.


Correlates of behavior problems in children with epilepsy.

We report results from the first part of an ongoing longitudinal study aimed at identifying the relative contributions of demographic, seizure, and family variables in the prediction of behavior problems in children with epilepsy. We studied 127 children with epilepsy aged 8-12 years and their mothers. Self-report questionnaires, interviews, and medical records were data sources. Backward and forward stepwise elimination procedures using multiple regression indicated five variables that contributed significantly to prediction of behavior problems: female gender, family stress, family mastery, extended family social support, and seizure frequency. These factors accounted for 29% (p < 0.001) of the variation in behavioral problems. Findings suggest that family variables are important correlates of behavior problems and should be considered in clinical management of children with epilepsy.


Bilateral Todd's paralysis after focal seizures.

Transient postictal hemiparesis or monoparesis is not uncommon after partial (focal) seizures. We report 2 patients who complained of severe bilateral limb weakness after generalized tonic-clonic seizures (GTCS) beginning focally. Bilateral Todd's paralysis was verified and documented in both patients. EEG and clinical evidence indicate the supplementary motor cortex as the most likely source of the seizures in both cases.


Psychosocial outcomes of antiepileptic drug discontinuation. The Medical Research Council Antiepileptic Drug Withdrawal Study Group.

The MRC Antiepileptic Drug Withdrawal Study compared seizure control and the risk of relapse resulting from policies of slow discontinuation and routine maintenance of antiepileptic drug (AED) treatment in patients seizure-free for > or = 2 years. Because the decision to discontinue AEDs can have important psychosocial as well as medical consequences, we also sought to examine the psychosocial outcomes of the alternative policies Questionnaires were sent to eligible adults 2 years after their randomization. The response rate was 85%. There was little evidence of substantial effects of treatment policy on psychosocial outcomes, but seizure recurrence since randomization was associated with increased distress on several measures. However, receiving AEDs to control seizures was also associated with increased distress. Among patients for whom the risk of relapse after discontinuation appears low, the psychosocial benefits of discontinuation may be considerable.


Efficacy of the ketogenic diet for intractable seizure disorders: review of 58 cases.

The ketogenic diet was developed in the 1920s as a treatment for intractable childhood seizures when few antiepileptic drugs (AEDs) were available. There are still children whose seizures are refractory even to modern therapy, but use of the ketogenic diet appears to be waning. At Johns Hopkins, we continue to believe that the diet is very effective and well accepted by patients and families. To reevaluate our opinion of the efficacy and acceptability of this form of therapy in patients cared for in the 1980s with the newer AEDs, we analyzed the records of 58 consecutive patients who had been started on the diet. Before using the diet, 80% of the patients had multiple seizure types and 88% were treated with multiple AEDs; these children were among our most intractable patients. Despite this, seizure control improved in 67% of patients with the ketogenic diet, and actuarial analysis indicated that 75% of these improved patients continued the diet for at least 18 months. Sixty-four percent had AEDs reduced, 36% became more alert, and 23% had improved behavior. The improvement in these patients with intractable seizures and the length of time that families maintained the regimen indicate that the ketogenic diet continues to have a very useful therapeutic role in selected patients and their families.


Oxcarbazepine does not affect the anticoagulant activity of warfarin.

The possible interaction of the antiepileptic drug oxcarbazepine (OCBZ) on the anticoagulant effect of warfarin was investigated in 10 healthy male volunteers. After reaching steady-state conditions by repeated administration of warfarin, the prothrombin time (Quick value) was assessed before and after single (600 mg) and multiple dosing (450 mg twice daily in 1 week) of OCBZ. In 7 of the 10 volunteers with evaluable data, the prothrombin time was not significantly different (paired t test) from baseline either after single (p = 0.299) or repeated dosing (p = 0.333), indicating that OCBZ does not interact to any relevant extent with the hypothrombinemic effect of warfarin.


Carbamazepine (CBZ) controlled release compared with conventional CBZ: a controlled study of attention and vigilance in children with epilepsy.

To compare the effects on attention and vigilance of conventional carbamazepine (CBZ) and CBZ controlled release (CBZ-CR), 15 schoolchildren with epilepsy and normal intelligence receiving CBZ were switched to CBZ-CR. Psychological examination was performed on the day before (day A) and 1 month after substitution (day B). Measurements of attention and vigilance were repeated throughout the day at 2-h intervals. Both on days A and B, CBZ plasma levels were monitored during a 12-h period. Fifteen matched healthy controls were submitted to identical test programs, allowing comparisons within subjects as well as between patients and control children. Variability of performance over sessions was neither increased nor decreased with CBZ-CR. Consistent differences over sessions did not exist between patients and controls with either drug. No relation was noted between reaction time and CBZ plasma level. The pharmacologic data confirm results of earlier studies in children. We noted a reduction in intradose fluctuations of CBZ level with CBZ-CR as compared with conventional CBZ. The neuropsychological results do not show a difference between the drugs. Neither were differences noted with respect to antiepileptic efficacy and side effects. At the end of the study, all but one of the children (and their parents) opted to receive CBZ-CR for ease of dosage administration and overall satisfaction.


Defining lead as the premiere environmental health issue for children in America: criteria and their quantitative application.

The principal environmental health issue for American children is pervasive lead poisoning from the many decades of lead contamination. Available scientific evidence cementing lead's premiere ranking is voluminous, multifaceted, and compelling. This evidence, however, requires organization into a clear and coherent body of science before it can be fully recognized or comprehended by either the scientific community or the general public and its representatives: public health officials, regulators, policy makers, and legislators. An attempt at such organization is presented and begins with the premise that there exist clear, objective criteria by which a premiere environmental health issue can be defined. A second premise is that these criteria sort themselves into three categories which cover the full spectrum of toxic contaminant-population relationships. They are: (1) economic and sociopolitical, (2) scientific and public health, and (3) societal risk assessment criteria. The first set of criteria includes economic and historical centrality, primacy of economic over public health considerations, a relatively narrow decision-making framework, and controlled flow of information on the toxicant, especially its negative impacts. The second set of criteria is also orthodox in scope: the toxicant should be indestructible, should accumulate in both the environment and the body, and should be a multimedia contaminant; it should produce toxicity in numerous organs with little impediment; toxicity should be produced with low/no threshold in huge numbers of the most vulnerable; and finally, effects should persist in the critical target organ(s). There is a third, more globally encompassing, set of criteria important for present-day requirements for risk assessment; e.g., the contaminant should produce full-spectrum population-wide as well as individual toxicity. Evidence for societal harm should be compelling. It should typify the increasing importance of the elements of preventive over clinical medicine and the substance should bring to bear the cost-benefit analysis of macro plus micro health risk. Lead exposure and toxicity is conclusively shown to meet ALL of these criteria and is the premiere environmental health threat to America's children.


Possible interaction between oxcarbazepine and an oral contraceptive.

The effect of oxcarbazepine (OCBZ) on the kinetics of an oral contraceptive containing ethinyloestradiol (EE) and levonorgestrel (LNG) was investigated in 13 healthy female volunteers who had previously received the contraceptive for at least 3 months. After 15 days of the first study cycle, each subject received, in addition to the oral contraceptive, 300 mg OCBZ on day 16, 300 mg twice daily on day 17, and 300 mg three times daily from day 18 of the first cycle to day 18 of the next menstrual cycle. The area under the curve values for both EE and LNG decreased when OCBZ was given with the oral contraceptive (p = 0.006, analysis of variance). The results indicate that OCBZ, like most antiepileptic drugs (AEDs), decreases the bioavailability of EE and LNG, perhaps by affecting metabolism or protein binding.


N-methyl-D-aspartate injection into the massa intermedia facilitates development of limbic kindling in rats.

The effect on limbic kindling of N-methyl-D-aspartate (NMDA) injection into the midline thalamus of rats was investigated. Repeated injection of 25 nmol/0.5 microliters NMDA (experimental group) into the massa intermedia (MI), particularly into the reuniens nucleus, caused tonic and/or clonic generalized convulsion associated with temporal limbic EEG seizure discharge. This did not occur after injection of phosphate-buffered saline (PBS, control group). When the animals were subjected to subsequent kindling at either the hippocampus (HP) or the amygdala (AM), only the experimental group showed a significant facilitation of kindling rate. The results suggest that an NMDA receptor in the reuniens nucleus participates in modulation of temporal limbic excitability and seizure development.


Screening for lead exposure using a geographic information system.

Screening programs for lead overexposure typically target high-risk populations by identifying regions with common risk markers (older housing, poverty, etc.). While more useful than untargeted screening programs, targeted programs are limited by the geographic resolution of the risk-factor information. A geographic information system can make screening programs more effective and more cost-efficient by mapping cases of overexposure, identifying high-incidence neighborhoods warranting screening, and validating risk-factor-based prediction rules.


Action of antiepileptic drugs against kainic acid-induced seizures and automatisms during ontogenesis in rats.

Kainic acid (KA 4-14 mg/kg) administered intraperitoneally (i.p.) produces automatisms (scratching until third postnatal week, "wet dog" shakes thereafter), and clonic and tonic-clonic seizures in rats aged 7, 12, 18, 25, and 90 days. Administration of carbamazepine (CBZ) i.p. (25 or 50 mg/kg), phenobarbital (PB 20-80 mg/kg), clonazepam (CZP 0.2 or 1 mg/kg), or valproate (VPA 200 mg/kg) influenced neither incidence nor latency of automatisms. Clonic seizures that are regularly observed after the third postnatal week in controls were either abolished or substantially suppressed by any of the aforementioned antiepileptic drugs (AEDs). Tonic-clonic seizures observed in the first 3 postnatal weeks were suppressed only by solvent [including propyleneglycol (PEG), ethanol, and water]; the effect of AEDs on tonic-clonic seizures was proconvulsant instead. The automatisms were most resistant to AED therapy. These results induce some doubts about the adequacy of the KA model for identifying AEDs effective against complex partial seizures, but forthcoming AEDs that suppress automatisms in the KA rat model might also be active against human complex partial seizures.


Reassessment of lead exposure in New Jersey using GIS technology.

In order to prevent children's exposure to lead, a variety of sources must be controlled. The New Jersey Department of Environmental Protection and Energy (NJDEPE) is using its Geographic Information System to identify areas within Newark, East Orange, and Irvington, New Jersey, where there may be greater environmental exposure to lead. Sensitive populations are identified through the U.S. Bureau of the Census information. Blood screening data provided by the New Jersey Department of Health (NJDOH) provide reported patterns of elevated blood lead in the study area. Comparisons of these spatial patterns will assist the NJDEPE in its soil sampling activities and lead exposure research, will provide information for public education, and will provide valuable information on sections of the study area where further screening and public education may be needed.


In vivo X-ray fluorescence of lead in bone.

The in vivo measurement of lead in bone by K X-ray fluorescence (K XRF) is becoming an increasingly widely utilized technique for assessing long-term lead dosimetry. Several groups have already reported the development of in vivo measurement systems, the majority adopting the 109Cd/backscatter K XRF technique because of its substantial advantages in terms of a robust measurement, lower detection limit (compared with 57Co/90 degrees), absence of the need for sedation of younger subjects, and lower effective (radiation) dose when calculated according to the most recent guidelines. The advantages of the 109Cd system are primarily a consequence of the physics principles of the technique. The apparatus of each XRF technique is transportable, facilitating easy establishment of mobile laboratory facilities. More research is needed to substantiate claims made for particular XRF technologies, but both L and K XRF techniques provide the possibility of improved understanding of the body's handling of the ubiquitous toxin lead.


Age-dependent changes in long-term seizure susceptibility and behavior after hypoxia in rats.

We showed that hypoxia is acutely epileptogenic in immature but not in adult rats. In the present study, we evaluated whether hypoxia results in an increase in long-term seizure susceptibility to flurothyl and whether this is associated with impaired performance on behavioral tests. We also determined whether these long-term outcomes are dependent on age at time of O2 deprivation. Long Evans hooded rats were rendered hypoxic on either postnatal day (P)5, P10, or P60. Sixty to 75 days after hypoxia, rats were tested for performance in water maze, open field, and handling tests and for seizure susceptibility to flurothyl. Hypoxia at P10 significantly increased seizure susceptibility to flurothyl, whereas hypoxia at P5 and P60 induced no long-term changes in seizure threshold. At P10, greater seizure severity during hypoxia and more prolonged exposure to hypoxia significantly increased long-term seizure susceptibility. This long-term change in seizure susceptibility appeared to be dissociated from any long-term neurobehavioral consequences, because only animals rendered hypoxic as adults (P60) had impaired behavioral performance. The results suggest that hypoxia-induced seizures can alter long-term seizure susceptibility and that this long-term effect is dependent on age and on severity of seizure activity at the time of previous hypoxia.


Anticonvulsant effect of DN-1417, a derivative of thyrotropin-releasing hormone, and liposome-entrapped DN-1417, on amygdaloid-kindled rats.

The effects of gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-propionamide citrate (DN-1417), a derivative of thyrotropin-releasing hormone, and liposome-entrapped DN-1417 (DN-L) were examined in amygdaloid-kindled rats. The animals were tested 2 h after intraperitoneal (i.p.) drug administration and then again every 24 h without further drug treatment. DN-1417 did not suppress the kindled seizure at 2 h but did beginning 1-6 days after injection. DN-L suppressed the kindled seizure at 2 h and had a more prolonged anticonvulsant effect than DN-1417. After liposomes were given i.p. once daily for 2 weeks, there was no morphologic evidence that liposomes damaged brain neurons. These results, together with previously published data, suggest that as drug delivery vehicles, liposomes can enhance the effectiveness of drugs in the CNS without producing overt brain damage.


Immunological findings in hemp workers.

Immunological status and its relation to respiratory findings were studied in 42 female textile workers occupationally exposed to hemp dust and in 49 female control workers. Skin prick tests with hemp or flax dust extracts from different parts of the mill in hemp workers demonstrated the following frequencies of positive tests to antigens: a mixture of hemp and flax extracts (64%), followed by flax extracts (48%), hemp from combing machines (41%), hemp from carding machines (38%), hemp from spinning and weaving machines (33%), and hemp from softening machines (20%). The prevalence of positive skin tests to hemp or flax allergens in control workers was consistently lower, ranging from 21 to 5%. Increased total serum IgE was recorded in 35.7% of hemp workers compared to only 5.0% of control workers (P < 0.05). Hemp workers with positive skin tests had significantly higher prevalences of chronic respiratory symptoms than those with negative skin tests. There were, however, no differences for acute symptoms between workers with positive and negative skin tests. Across-shift changes and baseline lung function were not different when compared by immunologic status. We showed additionally that a water-soluble extract of hemp dust causes a dose-related contraction of nonsensitized guinea pig tracheal smooth muscle when studied in vitro. Our results suggest that frequent immunologic abnormalities can be documented in hemp workers but, with the exception of chronic respiratory symptoms, in general, these do not correlate with respiratory findings.


Air pollution and daily mortality: associations with particulates and acid aerosols.

The association between total daily mortality and air pollution was investigated for a 1-year period (September 1985 through August 1986) in St. Louis and in the counties in eastern Tennessee surrounding Kingston/Harriman. The purpose of this study was to evaluate the relative importance of various measures of particulate and gaseous air pollution as predictors of daily mortality. Concentrations of inhalable particles (PM10), fine particles (PM2.5), the elemental composition of these particles, and aerosols acidity were measured daily during the period of study. The effect of each air pollutant on daily mortality was estimated after controlling for meteorologic and seasonal influences. Total mortality in St. Louis was found to increase 16% (95% CI-1 to 33%) for each 100 micrograms/m3 increase in PM10, and by 17% (95% CI-12 to 57%) in eastern Tennessee. Positive but progressively weaker associations were found with PM2.5, sulfate, and aerosol acidity concentrations in both communities. Associations with gaseous pollutants--sulfur dioxide, nitrogen dioxide, and ozone--were all far from statistical significance. Because of the short monitoring period for daily particulate air pollution, the power of this study to detect associations was limited. Nevertheless, statistically significant associations with PM10 were found in St. Louis, and, more importantly, the estimated effects were consistent between the two communities studied and with other reported analyses of the effects of particles on daily mortality. These data suggest that the acidity of particles is not as important in associations with daily mortality as the mass concentrations of particles.


Temporal patterns in air pollution and hospital admissions.

A 6-year data set of daily counts of admissions to 79 acute care hospitals in Southern Ontario was analyzed in relation to concurrent measurements of air pollution and weather pooled over the same regions, using progressively more sophisticated statistical techniques. The diagnoses studied included a group of respiratory causes and two control diagnoses: accidents and gastrointestinal causes. The 6-year period (1979-1985) was subdivided into six 2-month "seasons" and the area of study was divided into three subregions. Bivariate correlations were found to be significant more often than expected due to chance for all three admissions variables, but accounting for the temporal variation within the 60-day seasons greatly reduced the significance of the control diagnoses. Twenty-four-hour averages for air quality were found to yield more significant associations than peak hourly concentrations. July-August was the only period not having important within-season temporal trends and also had the lowest daily counts for respiratory admissions. Based on a model which accounted for serial correlation, SO2, ozone, and sulfate aerosol were found to be significant predictors of respiratory admissions during July-August. Using cumulative lags increased the magnitude of the estimated response to about 20% of summer respiratory admissions, but no consistent relationships were found which could identify the "responsible" pollutant(s) with certainty. Average pollutant concentrations were generally within U.S. ambient standards.


Beryllium-induced lung disease in the dog following two exposures to BeO.

We have shown previously that dogs exposed once to aerosols of beryllium oxide (BeO) calcined at 500 or 1000 degrees C developed granulomatous lung lesions as well as Be-specific immune responses in the blood and lung. In this report, we investigate the immunopathologic consequences of exposing dogs twice to aerosols of BeO. Dogs previously exposed to aerosols of 500 or 1000 degrees C calcined BeO to achieve an initial lung burden (ILB) of either 50 or 17 micrograms/kg body wt were exposed a second time to BeO calcined at 500 degrees C, 2.5 years after the first exposure, to achieve an ILB of about 50 micrograms/kg body wt. Immune responses of peripheral blood and lung lymphocytes were measured at 0, 14, 30, 60, 90, 120, 150, 165, 180, and 210 days postexposure (dpe), and dogs were euthanized at 210 dpe. Be-specific immune responses occurred in blood at 30 dpe and again at 150 to 210 dpe. Only sporadic positive responses were seen among lung lymphocytes when cells were cultured in 10% fetal bovine serum. In contrast, samples collected at 165, 180, and 210 dpe and incubated with 10% dog serum showed a large number of positive responses in both blood and lung. Histologic lesions were characterized by perivascular and interstitial infiltrates of lymphocytes and macrophages with progression to patchy granulomatous pneumonia accompanied by focal septal fibrosis. We conclude that Be-induced granulomatous and fibrotic lung lesions are accompanied by Be-specific immune responses within the lung but these changes do not appear to be cumulative if enough time has elapsed between exposures.


Dose-dependent pulmonary effects of inhaled endotoxin in guinea pigs.

As a cell wall component of gram-negative bacteria, endotoxin is thought to play a significant role in the respiratory effects of inhaled organic dusts which are microbially contaminated. Assessment of occupational survey data and clinical studies suggests that few measureable, acute functional changes occur below 30-50 ng/m3 endotoxin (as sampled in airborne dust with a vertical elutriator). Little information is available on the inflammatory effects of inhaled endotoxin at these low concentrations. The present study examined the dose-response relationship between inhaled endotoxin and functional, biochemical, and histological endpoints in the lungs of guinea pigs. Animals were exposed to 0.03 to 50.5 micrograms/m3 aerosolized endotoxin or the vehicle water for 4 hr. At 2 hr into exposure, significant decreases in specific airway conductance were observed only in animals exposed to 9.6 and 50.5 micrograms/m3 endotoxin (17.3 +/- 1.2 and 35.5 +/- 0.5% decreases from baseline values, respectively (mean +/- SE)). Total cell count and lactate dehydrogenase levels in bronchoalveolar lavage fluid were significantly elevated at 24 hr after exposure in all endotoxin-exposed groups except the lowest dose, 0.03 micrograms/m3 (P < 0.05). Polymorphonuclear leukocyte influx into the alveolar region was also dependent on the concentration of inhaled endotoxin. Thus, LDH activity, a biochemical marker of cell injury, and total cell counts and polymorphonuclear leukocytes, markers of inflammation, were more sensitive indices of adverse pulmonary effects from inhaled endotoxin than a functional measurement. These results suggest that subtle inflammatory changes may occur at airborne endotoxin concentrations which may produce no acute respiratory symptoms.


Assessment of slow axonal transport in lead-exposed rats.

To evaluate the effect of lead on slow axonal transport, mean transport velocity (Vmean) of radiolabeled proteins in the motor fibers of the sciatic nerve was calculated by simulating the distribution of radioactivity along the nerve 2 weeks after intraspinal injection of L-[35S] methionine in eight rats given drinking water containing lead acetate for 13 weeks. The transport of individual radiolabeled proteins was also inspected visually by fluorography. It was observed that the Vmean was significantly slowed in the lead-exposed rats and was correlated with blood lead concentration; fluorography indicated that the transport of neurofilament proteins and of tubulins was mildly retarded in the most severely affected lead-exposed rat. These findings suggest that slow axonal transport was impaired by lead. Further study using a large number of animals will be necessary to confirm the findings of the present study.


Effects of deltamethrin on the immune system in mice.

The influence of deltamethrin on the immune system in mice was investigated. Female BALB/c mice received deltamethrin in two daily oral doses; 6 mg/kg for 84 days and 15 mg/kg for 14 days. The humoral immune response in animals immunized with sheep red blood cells determined by agglutinin and hemagglutinin titer as well as by the number of plaque-forming cells producing IgM antibodies was significantly decreased. Also, the cell-mediated immune response assessed by alpha-naphthyl acetate esterase activity, by formation of EAC (erythrocyte-antibody-complement) rosettes, and by the footpad reaction test was hindered. A decrease in interleukin-1 activity was also ascertained. The obtained results indicate that deltamethrin exhibits an immunosuppressive effect.


Detection of DNA damage in stimulated human lymphocytes after enflurane exposure in vitro.

DNA damage was detected by nucleoid sedimentation in human lymphocytes stimulated with pokeweed mitogen after exposure to enflurane. Enflurane induces DNA damage at an exposure concentration of 0.2 vol%. Higher enflurane concentrations increase the rate of DNA damage. The DNA damage seen after exposure to enflurane concentrations of 0.2 and 3.0% vol is comparable to damage after X-radiation of 0.1 and 0.7 Gy. DNA single-strand breaks can be demonstrated by nucleoid sedimentation and can indicate damage before DNA repair begins. Therefore, detected DNA single-strand breaks may be reversible. However, DNA repair is not always successful and an increased number of DNA single-strand breaks could lead to irreversible DNA damage. The method of nucleoid sedimentation helps to show DNA damage in proliferating cells after exposure to volatile anesthetics or therapeutic gases.


Effects of zonisamide on extracellular levels of monoamine and its metabolite, and on Ca2+ dependent dopamine release.

The effects of zonisamide (3-sulfamoylmethyl-1,2-benzisoxazole), a novel anticonvulsant, on extracellular levels of monoamine and its metabolite in the striatum and hippocampus, and Ca2+ dependent monoamine release in the striatum of freely moving rats were studied by microdialysis. Zonisamide increased dopamine, homovanillic acid and 5-hydroxyindoleacetic acid, and decreased 3,4-dihydroxyphenylacetic acid in the rat striatum. However, zonisamide showed no effect on Ca2+ dependent dopamine release in the rat striatum. In the hippocampus, zonisamide increased dopamine, homovanillic acid, serotonin and 5-hydroxyindoleacetic acid and decreased 3,4-dihydroxyphenylacetic acid. The present results suggest that zonisamide facilitates dopaminergic and serotoninergic neurotransmission but does not affect Ca2+ dependent dopamine release within therapeutic plasma concentrations.


Neurovascular microdysgenesis in a case of Unverricht-Lundborg's disease.

An 18 year old female presented with a characteristic clinical course of Unverricht-Lundborg's disease. She was treated with several antiepileptic drugs but never with phenytoin. This patient died quietly during sleep. Coronal sectioning of the fixed brain revealed a 10 mm large red-brown solid cortical-subcortical lesion in the mesial part of the right frontal lobe and a similar alteration measuring 4 mm in the centromedian nucleus of the left thalamus. Histologically the lesions displayed supernumerary irregularly oriented vessels, mainly capillaries, and, in the cortex, a markedly disturbed neuronal orientation and lamination. Numerous heterotopic nerve cells were found in the subcortical white matter, especially under the calcarine cortex. A similar vascular malformation was found in the pons. The neuropathological changes in Unverricht-Lundborg's disease have been described as non-specific atrophy of various neuronal populations, especially medial thalamic nuclei and Purkinje cells. The present case shows that the same clinical picture may be associated with multiple cerebral microvascular malformations and a disturbance in neuronal migration and organization which may be more generalized than appears in light microscopical sections.


Carbamazepine induced bradycardia--a problem in general or only in susceptible patients? A 24-h long-term electrocardiogram study.

Carbamazepine is a first line drug in the treatment of epilepsy and trigeminal neuralgia, but may exert negative chronotropic and dromotropic effects on the cardiac conduction system. Bradyarrhythmias of different types and severity have been described, especially in the elderly, but the prevalence of arrhythmias in a larger group of carbamazepine treated patients is unknown. Forty-eight patients, 40 years of older, on continuous carbamazepine treatment because of various neurologic disorders were investigated by interview, physical examination, 12-lead surface electrocardiogram, and 24-h long-term electrocardiogram recording. The prevalence of bradyarrhythmias was compared with that in an age-stratified reference group. There was no differences between the two groups, either in the number or the duration of pauses or in the type of pauses. In conclusion, carbamazepine does not increase the risk of bradyarrhythmias in the vast majority of patients.


Protein binding of four antiepileptic drugs in maternal and umbilical cord serum.

The total and protein free levels of 4 antiepileptic drugs (AEDs) in serum from 35 maternity patients who had been treated with AED monotherapy throughout pregnancy were studied. Results were compared with those in the umbilical cord serum at the time of delivery, and the placental transfer of AEDs was evaluated from the viewpoint of the protein binding capacity of the drug. The materials consisted of 35 samples of maternal and umbilical cord serum in total and included 13 patients on phenobarbital (PB), 7 on phenytoin (PHT), 7 on carbamazepine (CBZ) and 8 on valproic acid (VPA). The mean fetal/maternal total concentration ratios were 0.86, 0.91, 0.73 and 1.59 for PB, PHT, CBZ and VPA, respectively, only the VPA ratio being above 1. On the other hand, the mean fetal/maternal free fraction ratios were 1.13, 1.10, 1.42 and 0.50 for PB, PHT, CBZ and VPA, respectively, only the VPA ratio being less than 1. Correlation of the 2 ratios showed a reciprocal proportion with a correlation coefficient of -0.90 (P < 0.005). It was considered that the fetal/maternal total concentration ratio of 4 AEDs was regulated by the fetal/maternal free fraction ratio of the corresponding AEDs and that the difference in fetal/maternal free fraction ratio depended on the type of drug being administered.


Comparison of PET measurements of cerebral blood flow and glucose metabolism for the localization of human epileptic foci.

We compared the relative sensitivity of two interictal PET techniques, bolus injection of [15O] labeled water for estimation of cerebral blood flow (H2(15)O CBF-PET), and 18F 2-deoxyglucose (18FDG-PET) for cerebral glucose metabolism (CMRglc), and T2-weighted magnetic resonance imaging, in 28 patients with medically intractable complex partial seizures undergoing evaluation for surgery. There were statistically significant associations between lateralization by 18FDG-PET, and MRI, but not H2(15)O CBF-PET, and lateralization of the epileptic focus as defined by scalp-sphenoidal ictal EEG. Fifteen patients had surgery or subdural electrodes. 18FDG-PET was more closely associated with a good outcome than H2(15)O CBF-PET, which, in addition, showed hypoperfusion contralateral to the epileptic temporal lobe in several cases. H2(15)O sensitivity may have been reduced by technical factors, but 18FDG-PET appears to be more specific for localization of epileptic zones.


Long-term treatment with gabapentin for partial epilepsy.

Gabapentin was studied as an open-label 'add-on' antiepileptic drug in 35 patients with partial seizures. Follow-up at 6 months, 12 months, 18 months, and 24 months is reported. There was a trend toward improvement in simple (SPS) and complex partial seizures with it reaching significance for SPS at 12 and 24 months and for the weighted combination of seizures at 3 months. Five of nine patients were subsequently successfully converted to gabapentin monotherapy. Of those five, one is now seizure free and three are significantly improved since baseline. One remains with unchanged seizure frequency compared to baseline, but is experiencing less toxicity than at that time. This long-term observation suggests that the short-term effect demonstrated in blinded studies continues and that indeed some patients with refractory epilepsy can be maintained on gabapentin alone. Based on these findings, double-blind monotherapy trials of this drug are presently being conducted.


The phenomenology of multiple partial seizure-like symptoms without stereotyped spells: an epilepsy spectrum disorder?

Consistent with previous reports by neuropsychiatrists, the results of the present investigation confirmed the existence of a neurobehavioral disorder characterized by the subjective experiencing of multiple cognitive, affective, and psychosensory phenomena similar to those associated with more classic partial seizure disorders. According to the literature, such patients typically respond favorably to anticonvulsants although they lack the customary motor manifestations of complex partial seizure (CPS) disorders and typically do not have stereotyped spells. This neuropsychiatric syndrome has recently been termed epilepsy spectrum disorder (ESD). In the present study, 30 patients with ESD were matched with equal numbers of treatment-refractory CPS patients and normal controls. All subjects were administered a standardized interview consisting of 35 cognitive, affective, and psychosensory partial seizure-like symptoms. The results indicated that ESD patients endorsed significantly more partial seizure-like symptoms than did CPS patients and controls. Relatively low levels of symptom endorsement by an unmatched psychiatric comparison sample indicated that the high levels of symptom endorsement by ESD patients could not be attributed to the presence of psychiatric dysfunction per se. Analysis of responding to 'foil' items unrelated to partial seizures indicated that high levels of symptom endorsement by ESD patients did not merely reflect a deviant response. Although ESD patients seldom present themselves at tertiary care epilepsy centers, the study of such patients is likely to be of relevance to mainstream epileptology.


Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus.

We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabidopsis thaliana. The predicted product shares no homology to known sequences. ELI3 mRNA accumulates in A. thaliana leaves in response to challenge with phytopathogenic Pseudomonas syringae strains. The timing and magnitude of this response are dictated by the genetics of the plant-pathogen interaction being analyzed. During incompatible interactions, where resistance in the plant genotype Col-0 is dictated by the dominant RPM1 locus, ELI3 mRNA accumulates to high levels 5-10 h post-inoculation. This kinetic behavior is also generated by the presence of a cloned bacterial avirulence gene, in otherwise virulent bacteria, which triggers resistance mediated via RPM1 action. The phenotypic outcome is a hypersensitive resistance reaction visible 8-15 h post-infiltration. Thus, the induction kinetics of ELI3 mRNA accumulation are consistent with a functional role for the ELI3 gene product in establishing the resistant phenotype. In contrast, during compatible interactions with the susceptible plant genotype Nd-0, which is homozygous recessive at the rpm1 locus, ELI3 mRNA accumulates significantly only after 15 h. We show genetically that ELI3 activation is strictly dependent on the presence of dominant alleles at RPM1 using an assay generalizable to any pathogen induced plant defense phenomena.


Chilling sensitivity of Arabidopsis thaliana with genetically engineered membrane lipids.

Upon transfer of a genetically engineered Escherichia coli gene for glycerol-3-phosphate acyltransferase (plsB) to Arabidopsis thaliana (L.) Heynh., the gene is transcribed and translated into an enzymatically active polypeptide. This leads to an alteration in fatty acid composition of membrane lipids. From these alterations it is evident that the enzyme is located mainly inside the plastids. The amount of saturated fatty acids in plastidial membrane lipids increased. In particular, the fraction of high-temperature melting species of phosphatidylglycerol is elevated. These molecules are thought to play a crucial role in determining chilling sensitivity of plants. An increase in sensitivity could be observed in the transgenic plants during recultivation after chilling treatment. Implications for the hypothesis of phosphatidylglycerol-determined chilling sensitivity are discussed.


Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast.

Active loading of the phloem with sucrose in leaves is an essential part of the process of supplying non-photosynthetic tissues with carbon and energy. The transport is protein mediated and coupled to proton-symport, but so far no sucrose carrier gene has been identified. Using an engineered Saccharomyces cerevisiae strain, a cDNA from spinach encoding a sucrose carrier was identified by functional expression. Yeast strains that allow the phenotypic recognition of a sucrose carrier activity were constructed by expressing a cytoplasmic invertase from yeast, or the potato sucrose synthase gene, in a strain unable to transport or grow on sucrose due to a deletion in the SUC2 gene. A spinach cDNA expression library established from the poly(A)+ RNA from source leaves of spinach and cloned in a yeast expression vector yielded transformed yeast clones which were able to grow on media containing sucrose as the sole carbon source. This ability was strictly linked to the presence of the spinach cDNA clone pS21. Analysis of the sucrose uptake process in yeast strains transformed with this plasmid show a pH-dependent uptake of sucrose with a Km of 1.5 mM, which can be inhibited by maltose, alpha-phenylglucoside, carbonyl cyanide m-chlorophenylhydrazone and p-chloromercuribenzenesulfonic acid. These data are in accordance with measurements using both leaf discs and plasma membrane vesicles from leaves of higher plants. DNA sequence analysis of the pS21 clone reveals the presence of an open reading frame encoding a protein with a molecular mass of 55 kDa. The predicted protein contains several hydrophobic regions which could be assigned to 12 membrane-spanning regions.(ABSTRACT TRUNCATED AT 250 WORDS)


Chimeric myosin regulatory light chains identify the subdomain responsible for regulatory function.

Regulatory light chains, located on the 'motor' head domains of myosin, belong to the family of Ca2+ binding proteins that consist of four 'EF-hand' subdomains. Vertebrate regulatory light chains can be divided into two functional classes: (i) in smooth/non-muscle myosins, phosphorylation of the light chains by a calcium/calmodulin-dependent kinase regulates both interaction of the myosin head with actin and assembly of the myosin into filaments, (ii) the light chains of skeletal muscle myosins are similarly phosphorylated, but they play no apparent role in regulation. To discover the basis for the difference in regulatory properties of these two classes of light chains, we have synthesized in Escherichia coli, chimeric mutants composed of subdomains derived from the regulatory light chains of chicken skeletal and smooth muscle myosins. The regulatory capability of these mutants was analysed by their ability to regulate molluscan myosin. Using this test system, we identified the third subdomain of the regulatory light chain as being responsible for controlling not only the actin-myosin interaction, but also myosin filament assembly.


G- to F-actin modulation by a single amino acid substitution in the actin binding site of actobindin and thymosin beta 4.

The actin binding sites of actobindin and thymosin beta 4, two small polypeptides that inhibit actin polymerization by interacting with monomeric actin, have been localized using peptide mimetics. Both sites are functionally similar and extend over 20 residues and are located in the NH2-terminus of the polypeptides. They can be dissected into two functional entities: a conserved hexapeptide motif (LKHAET or LKKTET), which forms the major contact site through electrostatic interactions with actin, and a non-conserved NH2-terminal segment preceding the motif, which exerts the inhibitory activity on actin polymerization probably by steric hindrance. The introduction of a glutamic acid at the third position in the motif, creating LKEAET or LKETET sequences, which are similar to those found in some F-actin binding proteins, converts the peptide's inhibitory phenotype into an F-actin stimulatory property. These results allow the proposal of a simple model for G- to F-actin modulation.


The mouse 5HT5 receptor reveals a remarkable heterogeneity within the 5HT1D receptor family.

Serotonin (5-HT) is a neuromodulator that mediates a wide range of physiological functions by activating multiple receptors. Using a strategy based on amino acid sequence homology between 5-HT receptors that interact with G proteins, we have isolated a cDNA encoding a new serotonin receptor from a mouse brain library. Amino acid sequence comparisons revealed that this receptor was a distant relative of all previously identified 5-HT receptors; we therefore named it 5HT5. When expressed in Cos-7 cells and NIH-3T3 cells, the 5HT5 receptor displayed a high affinity for the serotonergic radioligand [125I]LSD. Surprisingly, its pharmacological profile resembled that of the 5HT1D receptor, which is a 5-HT receptor subtype which has been shown to inhibit adenylate cyclase and which is predominantly expressed in basal ganglia. However, unlike 5HT1D receptors, the 5HT5 receptor did not inhibit adenylate cyclase and its mRNA was not found in basal ganglia. On the contrary, in situ hybridization experiments revealed that the 5HT5 mRNA was expressed predominantly in cerebral cortex, hippocampus, habenula, olfactory bulb and granular layer of the cerebellum. Our results therefore demonstrate that the 5HT1D receptors constitute a heterogeneous family of receptors with distinct intracellular signalling properties and expression patterns.


Multiple trimeric G-proteins on the trans-Golgi network exert stimulatory and inhibitory effects on secretory vesicle formation.

The role of heterotrimeric G-proteins on the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN) of PC12 cells was investigated. Using immunofluorescence and subcellular fractionation in conjunction with immunoblotting or ADP-ribosylation by either pertussis toxin or cholera toxin, TGN membranes were found to contain not only several alpha i/alpha o G-protein subunits including apparently alpha i3, but also alpha s. Pertussis toxin treatment of cells, which resulted in the stoichiometric ADP-ribosylation of alpha i/alpha o, a modification known to prevent their coupling to receptors, led to the stimulation of cell-free CSV and ISG formation, suggesting the presence of a guanine nucleotide exchange factor for alpha i/alpha o on the TGN. Mastoparan-7, a peptide known to mimic an activated receptor and to stimulate nucleotide exchange on alpha i/alpha o, inhibited cell-free vesicle formation, an effect abolished by pertussis toxin. In contrast, activation of alpha s by cholera toxin treatment of cells resulted in a stimulation of cell-free CSV and ISG formation. This stimulation could be reversed when the alpha subunits not activated by cholera toxin, i.e. alpha i/alpha o, were activated by GTP gamma S and [AIF4]-. Our results show that both inhibitory and stimulatory trimeric G-proteins on the TGN participate in the regulation of secretory vesicle formation.


Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling.

The STE4 gene, which encodes the beta subunit of the mating response G-protein in the yeast Saccharomyces cerevisiae, was subjected to a saturation mutagenesis using 'doped' oligodeoxynucleotides. We employed a genetic screen to select dominant-negative STE4 mutants, which when overexpressed from the GAL1 promoter, interfered with the signalling function of the wild type protein. The identified inhibitory amino acid alterations define two small regions that are crucially involved in transmitting the mating signal from G beta to downstream components of the signalling pathway. These results underline the positive signalling role of yeast G beta and assign for the first time the positive signalling function of a G-protein beta subunit to specific structural features.


The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components.

In the yeast Saccharomyces cerevisiae the G-protein beta gamma subunits have been shown to trigger downstream events of the pheromone response pathway. We have identified a new gene, designated STE20, which encodes a protein kinase homologue with sequence similarity to protein kinase C, which is required to transmit the pheromone signal from G beta gamma to downstream components of the signalling pathway. Overproduction of the kinase suppresses the mating defect of dominant-negative G beta mutations providing genetic evidence for an interaction with G beta, and epistasis experiments show that this kinase functions after or at the same point as G beta gamma, but before any of the other currently identified components of the signalling pathway. This points to a potentially new mechanism of G-protein mediated signal transduction, the activation of a protein kinase through G beta gamma.


Molecular cloning of human growth inhibitory factor cDNA and its down-regulation in Alzheimer's disease.

In previous studies, we discovered a growth inhibitory factor (GIF) that was abundant in normal human brain, but greatly reduced in Alzheimer's disease (AD) brain. Molecular cloning of a full-length cDNA for human GIF revealed that the GIF had striking homology to metallothioneins. Furthermore, it was determined that the GIF gene was on chromosome 16, as are the metallothionein genes. GIF, in contrast to metallothioneins, was found to be expressed exclusively in the nervous system. The GIF protein produced by Escherichia coli harboring the GIF cDNA in a prokaryotic expression vector inhibited the growth of neonatal rat cortical neurons. These results indicate that GIF is a new member of the metallothionein family with distinct tissue-specific expression and functions. Northern blot analysis revealed that expression of the GIF mRNA is drastically decreased in AD brains. The result raises the possibility that down-regulation of the GIF gene in AD brain plays an important role in the pathogenesis of AD.


Lineage-independent activation of immune system effector function by myeloid Fc receptors.

An emerging theme in immunology finds receptors which initiate cellular effector programs forming multichain complexes in which the ligand recognition elements associate with one or more 'trigger molecules' whose aggregation initiates a signal transduction cascade. The sequence motifs constituting the active sites of these trigger molecules are found in the T cell and B cell antigen receptors, and some Fc receptors, and appear to be central to effector function activation. For example, of the many molecules that mimic or potentiate the action of the T cell antigen receptor (TCR), none have yet been found to initiate effector programs autonomously in cells lacking TCR. We have devised two strategies to study activation mediated by myeloid Fc receptors, which appear not to associate with trigger molecules: the use of primary human cytolytic T cells as surrogate effector cells for genetically delivered receptors, and the use of vaccinia virus vectors to introduce genetically modified receptors into primary human monocytes. Using these approaches, we have found that the cytoplasmic domains of two Fc receptors show comparable function to equivalent domains of the trigger molecule family, but are not homologous to members of that family.


Biased reading frames of pre-existing DH--JH coding joints and preferential nucleotide insertions at VH--DJH signal joints of excision products of immunoglobulin heavy chain gene rearrangements.

During B cell differentiation immunoglobulin (Ig) DH segments join to JH segments, followed by joining of VH to DJH. Although circular excision products of DH--JH rearrangements have been characterized, excision products of VH to DJH joining have never been isolated. We selectively denatured chromosomal DNA of mouse splenocytes and enriched circular DNA spanning the long distance between VH and DH. Subsequent PCR amplifications allowed the identification of signal joints of VH to DJH. Sequence analysis indicated that preexisting DH--JH coding joints of excision products showed a strong bias for reading frame 1, and the absence of reading frame 2, which would allow the expression of a truncated mu chain called D mu protein. When comparing the joints of the VH--DJH and DH--JH rearrangements we observed N-nucleotide insertions to be abundant at the VH--DH signal joint, but very sparse at the DH--JH signal joint, while the coding joints of both contained abundant N-insertions. These differences in N region insertions at the signal joints suggest a differential control of the D--J and V--DJ rearrangements.


CD45 tyrosine phosphatase-activated p59fyn couples the T cell antigen receptor to pathways of diacylglycerol production, protein kinase C activation and calcium influx.

The role of the CD45 phosphotyrosine phosphatase in coupling the T cell antigen receptor complex (TCR) to intracellular signals was investigated. CD45- HPB-ALL T cells were transfected with cDNA encoding the CD45RA+B+C- isoform. The tyrosine kinase activity of p59fyn was found to be 65% less in CD45- cells than in CD45+ cells, whereas p56lck kinase activity was comparable in both sub-clones. In CD45- cells the TCR was uncoupled from protein tyrosine phosphorylation, phospholipase C gamma 1 regulation, inositol phosphate production, calcium signals, diacylglycerol production and protein kinase C activation. Restoration of TCR coupling to all these pathways correlated with the increased p59fyn activity observed in CD45-transfected cells. Co-aggregation of CD4- or CD8-p56lck kinase with the TCR in CD45- cells restored TCR-induced protein tyrosine phosphorylation, phospholipase C gamma 1 regulation and calcium signals. Receptor-mediated calcium signals were largely due (60-90%) to Ca2+ influx, and only a minor component (10-40%) was caused by Ca2+ release from intracellular stores. Maximal CD3-mediated Ca2+ influx occurred at CD3 mAb concentrations at which inositol phosphate production was non-detectable. These results indicate that CD45-regulated p59fyn plays a critical role in coupling the TCR to specific intracellular signalling pathways and that CD4- or CD8-p56lck can only restore signal transduction coupling in CD45- cells when brought into close association with the TCR.


Distinct downstream signaling mechanism between erythropoietin receptor and interleukin-2 receptor.

Erythropoietin receptor (EPOR) and interleukin-2 receptor beta chain (IL-2R beta) belong to the same cytokine receptor superfamily and have highly conserved sequences in their intracellular signaling domain. However, common downstream signaling pathways of these receptors have not been demonstrated. In the present study, we introduced and expressed the murine EPOR in murine IL-2-, IL-3- and IL-5-dependent cell lines and analyzed their growth response to EPO. We found that the expression of EPOR induced EPO dependence in IL-3-dependent BAF-B03 and IL-5-dependent Y16 cells but not in IL-2-dependent CTLL-2 cells, although the EPOR-expressing CTLL-2 cell lines could bind and internalize EPO as efficiently as the BAF-B03-derived cell lines. Additional expression of AIC2B, a common signal transducer for IL-3R, IL-5R and GM-CSFR, made no difference to the EPO responsiveness of the EPOR-expressing CTLL-2 cell lines. These results suggest that the cellular components required for the transduction of EPOR signal and IL-2R signal are at least partially different, and this difference cannot be explained solely by the absence of AIC2B.


A new cdc gene required for S phase entry of Schizosaccharomyces pombe encodes a protein similar to the cdc 10+ and SWI4 gene products.

We have isolated a new cell division cycle gene (res1+) required for entry into S phase, as a multicopy dual suppressor of the pat1 and cdc10 mutants of the fission yeast Schizosaccharomyces pombe. The res1+ gene specifies a 72 kDa protein with two copies of the cdc10/SWI6 motif. A disruptant of res1+ grows poorly at 30 degrees C with severe heat- and cold-sensitivities, and completely arrests in G1 at 36 degrees C and 23 degrees C. The arrested disruptant retains a full conjugation ability. In addition to the cdc10/SWI6 motif, Res1 and SWI4 proteins share a remarkable homology in their amino-terminal region, whereas Cdc10 and SWI6 do so in their carboxy-terminal region. Moreover, the amino-terminal region is essential for the function of Res1 as it is for the function of SWI4. Furthermore, analogous to the relationship of SWI4 to SWI6, the res1+ gene effectively rescues cdc10 mutants, but the cdc10+ gene cannot rescue the res1- phenotype. Thus, striking similarities exist in both structural and functional relationships between Res1 and SWI4, and between Cdc10 and SWI6. In view of the fact that SWI4 and SWI6 form a transcription factor complex and activate promoters containing the SWI4/SWI6 dependent cell-cycle box, Res1 might be a putative association partner of Cdc10 which appears to be involved at least in the activation of promoters containing a MluI cell-cycle box.


Pyp3 PTPase acts as a mitotic inducer in fission yeast.

The p34cdc2 M-phase kinase is regulated by inhibitory phosphorylation of Tyr15, largely through the actions of the p107wee1 tyrosine kinase and p80cdc25 protein tyrosine phosphatase (PTPase). In this study we demonstrate that a second PTPase, encoded by pyp3, also contributes to tyrosyl dephosphorylation of p34cdc2. Pyp3 was identified as a high copy suppressor of a cdc25- mutation. The pyp3 gene encodes a 33 kDa PTPase that is more closely related to human PTP1B and fission yeast pyp1 and pyp2 PTPases than to cdc25. Pyp3 does not share an essential overlapping function with pyp1 or pyp2. We demonstrate that disruption of pyp3 causes a mitotic delay that is greatly exacerbated in cells that are partially defective for cdc25 function and that pyp3 function is essential in cdc25-disruption wee1- strains. Pyp3 PTPase effectively dephosphorylates and activates the p34cdc2 kinase in vitro. We conclude that the pyp3 PTPase acts cooperatively with p80cdc25 to dephosphorylate Tyr15 of p34cdc2.


Negative regulation of mitosis by two functionally overlapping PTPases in fission yeast.

We have identified a third protein tyrosine phosphatase (PTPase) gene in fission yeast, pyp2, encoding an 85 kDa protein. Disruption of pyp2 has no impact on cell viability, but pyp2 is essential in strains lacking the 60 kDa pyp1 PTPase. The two pyp PTPases are approximately 42% identical in their C-terminal catalytic domains and share weak homology in their N-terminal regions. Both genes play a role in inhibiting the onset of mitosis. Disruption of either gene rescues the G2 arrest caused by mutation of the cdc25 mitotic inducer, though the effect of pyp1-disruption is more pronounced. Disruption of pyp1 advances mitosis, suppresses overexpression of the tyrosine kinase encoded by the wee1 mitotic inhibitor, and causes lethal mitotic catastrophe in cdc25 overproducer cells. Cells bearing inactive wee1 are unresponsive to disruption of pyp1. Overexpression of pyp1 or pyp2 delays the onset of mitosis by a wee1-dependent mechanism. These data reveal an unexpected second role for protein tyrosine phosphorylation in the mitotic control that acts by promoting the inhibitory wee1 pathway.


An S-phase specific release from a transcriptional block regulates the expression of mouse ribonucleotide reductase R2 subunit.

Ribonucleotide reductase (RR) activity in mammalian cells is closely linked to DNA synthesis. The RR enzyme is composed of two non-identical subunits, proteins R1 and R2. Both proteins are required for holoenzyme activity, which is regulated by S-phase specific de novo synthesis and breakdown of the R2 subunit. In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border. Using an R2 promoter-luciferase reporter gene construct we demonstrate an unexpected early activation of the R2 promoter as cells pass from quiescence to proliferation. However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear. The position for the transcriptional block was localized to a nucleotide sequence approximately 87 bp downstream from the first exon/intron boundary by S1 nuclease mapping of R2 transcripts from modified in vitro nuclear run-on experiments. These results identify blocking of transcription as a mechanism to control cell cycle regulated gene expression.


Different activation domains stimulate transcription from remote ('enhancer') and proximal ('promoter') positions.

We reported previously that the lymphocyte-derived octamer transcription factor 2A (Oct-2A or OTF-2A) activated both natural immunoglobulin promoters and synthetic promoters which contain the 'octamer' site, but was unable by itself to stimulate transcription from a remote enhancer position. Here we examine a larger set of transcription factors with respect to their proximal versus remote activation. Since a transcription factor may contain more than one activation domain, we have chosen to study the potential of individual activation domains in the context of fusion proteins that contain the DNA binding domain of GALA. We have identified at least two distinct functional classes of transcriptional activation domains. 'Proximal' activation domains, exemplified by glutamine-rich domains of Oct-1, Oct-2A and Sp1, stimulate transcription only from a position close to the TATA box, usually in response to a remote enhancer. 'General' activation domains, derived from VP16, GAL4, p65 (NF-chi B), TFE3, ITF-1 and ITF-2, can activate transcription from remote as well as proximal positions. These domains contain many acidic amino acids and/or other features such as clusters of serine and threonine. The proline-rich activation domains of AP-2 and CTF/NF1 may represent a third class with considerable promoter activity and low but significant enhancer activity. Furthermore, activation domains of both the acidic and glutamine-rich types seem to have a modular structure, since duplicated subdomains can substitute for the entire domain.


A synergistic increase in potency of a multimerized VP16 transcriptional activation domain.

Transcriptional synergy in eucaryotes provides a means to control both the level and diversity of gene expression. The mechanism by which multiple activators elicit such effects is unknown. To address this problem we considered whether multimerizing an activation domain was equivalent to oligomerizing an activator's binding sites on DNA. Synthetic activators bearing one, two or four VP16 'core' activation domains, fused to the GAL4 DNA binding domain, were co-transfected into Cos-1 cells with CAT gene reporter templates containing one, two or five upstream GAL4 binding sites. Our results demonstrate that all of the activators elicit synergistic effects when comparing the amounts of transcription on multiple sites versus a single site. In contrast, the multimerized activation domains did not stimulate transcription significantly on a template bearing a single site; a synergistic increase in potency was, however, apparent on a template bearing two sites. Introducing the flexible lambda repressor linker region in between the activation domains increased the ability of activators bearing two or four VP16 domains to stimulate transcription from the single-site template. We discuss the mechanistic implications of this study on gene activation and synergy.


The transcriptional transactivation function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6 oncoprotein.

The observed interaction between p53 and the oncoproteins encoded by several DNA tumor viruses suggests that these viruses mediate their transforming activities at least in part by altering the normal growth regulatory function of p53. In this study we examined the effect of viral oncoprotein expression on the transcriptional transactivation function of wild-type p53 in human cells. Plasmids expressing human p53 were cotransfected with either SV40 large T-antigen or human papillomavirus (HPV) type 16 E6 expression plasmids and assayed for transactivation function using a reporter gene driven by a p53-responsive promoter containing multiple copies of the consensus p53 DNA binding motif, TGCCT. Both large T-antigen and E6 were able to inhibit transactivation by wild-type p53. Furthermore, SV40 T-antigen mutants that are defective for p53 binding were not able to inhibit transactivation and HPV E6 proteins that were either mutant or derived from non-oncogenic HPV types and unable to bind p53, had no effect on p53 transactivation. These results demonstrate the physiological relevance of the interaction of SV40 T-antigen and HPV E6 oncoproteins with p53 in vivo and suggest that the transforming functions of these viral oncoproteins may be linked to their ability to inhibit p53-mediated transcriptional activation.


Polyadenylation of maternal mRNA during oocyte maturation: poly(A) addition in vitro requires a regulated RNA binding activity and a poly(A) polymerase.

Specific maternal mRNAs receive poly(A) during early development as a means of translational regulation. In this report, we investigated the mechanism and control of poly(A) addition during frog oocyte maturation, in which oocytes advance from first to second meiosis becoming eggs. We analyzed polyadenylation in vitro in oocyte and egg extracts. In vivo, polyadenylation during maturation requires AAUAAA and a U-rich element. The same sequences are required for polyadenylation in egg extracts in vitro. The in vitro reaction requires at least two separable components: a poly(A) polymerase and an RNA binding activity with specificity for AAUAAA and the U-rich element. The poly(A) polymerase is similar to nuclear poly(A) polymerases in mammalian cells. Through a 2000-fold partial purification, the frog egg and mammalian enzymes were found to be very similar. More importantly, a purified calf thymus poly(A) polymerase acquired the sequence specificity seen during frog oocyte maturation when mixed with the frog egg RNA binding fraction, demonstrating the interchangeability of the two enzymes. To determine how polyadenylation is activated during maturation, we compared polymerase and RNA binding activities in oocyte and egg extracts. Although oocyte extracts were much less active in maturation-specific polyadenylation, they contained nearly as much poly(A) polymerase activity. In contrast, the RNA binding activity differed dramatically in oocyte and egg extracts: oocyte extracts contained less binding activity and the activity that was present exhibited an altered mobility in gel retardation assays. Finally, we demonstrate that components present in the RNA binding fraction are rate-limiting in the oocyte extract, suggesting that fraction contains the target that is activated by progesterone treatment. This target may be the RNA binding activity itself. We propose that in spite of the many biological differences between them, nuclear polyadenylation and cytoplasmic polyadenylation during early development may be catalyzed by similar, or even identical, components.


A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis.

PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.


Chloroplast group III twintron excision utilizing multiple 5'- and 3'-splice sites.

The chloroplast genes of Euglena gracilis contain more than 60 group II and 47 group III introns. Some Euglena chloroplast genes also contain twintrons, introns-within-introns. Two types of twintrons have previously been described, a group II twintron and a mixed group II/group III twintron. We report that four introns, three within the RNA polymerase subunit gene rpoC1 and one within ribosomal protein gene rpl16, with mean lengths twice typical group III introns, are a new type of twintron. The group III twintrons are composed of group III introns within other group III introns. The splicing of the twintrons was analyzed by PCR amplification, cloning and sequencing of cDNAs, and Northern hybridization. Excision of each group III twintron occurs by a two-step, sequential splicing pathway. Removal of the internal introns precedes excision of the external introns. Splicing of internal introns in three of the four group III twintrons involves multiple 5'- and/or 3'-splice sites. With two of the twintrons the proximal 5'-splice site can be spliced to an internal 3'-splice site, yielding alternative 'pseudo' fully spliced mRNAs. Excised group III introns of the rpl16 twintron are not linear RNA molecules but either lariat or circular RNAs, probably a lariat. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.


A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1.

NSP1 is a nuclear pore protein (nucleoporin) essential for cell growth. To identify the components that functionally interact with NSP1 in the living cell, we developed a genetic screen for mutants that are lethal in a genetic background of mutated, but not wild type NSP1. Fourteen synthetic lethal mutants were obtained, belonging to at least four different complementation groups. The genes of two complementation groups, NSP116 and NSP49, were cloned. Like the previously described nucleoporins, these genes encode proteins with many repeat sequences. NSP116 and NSP49, however, contain a new repetitive sequence motif 'GLFG', which classifies them as a subclass of nucleoporins. NSP116 and NSP49, tagged with the IgG binding domain of protein A and expressed in yeast, are located at the nuclear envelope. These data provide in vivo evidence that distinct subclasses of nucleoporins physically interact or share overlapping function in nuclear pore complexes.