Iodide excretion before and after revision of goiter prophylaxis (Dutch Nutrition Surveillance System).

To assess the iodine supply in The Netherlands after the revision of the goiter prophylaxis measures (Bread Act of 1982, with an increase of iodine content of bread salt) the data of a nationwide survey among Dutch elderly people conducted in 1984/1985 were analysed. Iodide excretion in 24 h urine samples was used as the main iodine status indicator. The data were compared with data on iodine nutriture obtained among an elderly population in The Netherlands before the revision of goiter prophylaxis. High prevalences (greater than or equal to 37%) of low iodine excretion (less than 0.78 mumol/24 h; 100 micrograms/24 h) were found for Dutch elderly people. Mean urinary iodide excretion was 0.95 mumol/24 h (121 micrograms/24 h) for men and 0.79 mumol/24 h (100 micrograms/24 h) for women which is low, especially among women, in comparison with the United States recommended dietary allowance (118 mumol/day = 150 micrograms/day). Consistent positive associations of iodide excretion were found with urinary potassium and sodium excretion, bread consumption and total iodine intake. Bread, as the iodine carrier chosen for goiter prophylaxis in The Netherlands, was found to be the main dietary iodine source. No improvement in iodine nutrition was found among the elderly studied in 1984/1985 in comparison with an elderly population seen in 1981. Therefore, it is concluded that the present measures regarding goiter prophylaxis in The Netherlands might be of limited effectiveness.


Elevated plasma vitamers of vitamin B6 in patients with chronic renal failure on regular haemodialysis.

Plasma pyridoxal-5'-phosphate (PL-5'-P), pyridoxal (PL), pyridoxine (PN), and 4-pyridoxic acid (4-PA) were measured by high-performance liquid chromatography (HPLC) in 39 patients (15 male, 24 female) with chronic fenal failure undergoing regular haemodialysis and 46 healthy controls (28 male, 18 female). All three vitamers of vitamin B6 and the metabolite were significantly elevated in the haemodialysis patients. Mean PL-5'-P and PN concentrations were 20 times the mean in controls. Only one patient took a vitamin B6 supplement. In view of the neurotoxicity of supranutritional intakes of PN in normal humans we suggest that supplements of PN be carefully monitored when administered to patients with chronic renal failure.


I know distraction works even though it doesn't!

Resistance is high to findings negating commonsense beliefs. If McCaul, Monson, and Maki's (1992) four studies are taken seriously, we will address new questions about the components of analgesic interventions--specifically, whether distraction works only when combined with a competing affect, an analgesic cognition, or both. Addressing these questions should increase our understanding of the mechanisms involved in pain processing and may increase our ability to intervene and modify chronic as well as acute pain. Laboratory studies offer an efficient route to such understanding, although the question of generalization will always lurk in the background.


Does distraction reduce pain-produced distress among college students?

College students in four experiments placed their hands in ice water (the cold-pressor task) and reported their distress. They simultaneously engaged in different reaction-time (RT) tasks that varied in the amount of attention required for successful performance. In each experiment, which differed in numerous procedural details, RT, error-rate, and self-report measures all demonstrated that the distraction tasks differed in the degree of attention required. Greater distraction, however, failed to reduce physiological, self-report, or behavioral responses to the cold-pressor task. These data call into question the hypothesis that attention mediates the process whereby distraction tasks reduce pain-produced distress.


Stress, coping, and high-risk sexual behavior.

We examined the relation between stress, coping, and a high-risk sexual behavior (unprotected anal intercourse) in 398 nonmonogamous gay and bisexual men from the AIDS Behavioral Research Project in San Francisco. Unprotected anal intercourse during the previous month, the amount of stress experienced during the previous month in each of 10 domains, six types of coping (self-controlling coping, escape-avoidance, distancing, planful problem-solving, seeking social support, and positive reappraisal), and spiritual beliefs and spiritual activities were assessed through self-report. There was no relation between stress and unprotected anal intercourse. However, there was a relation between coping and unprotected anal intercourse. Subjects who reported unprotected anal intercourse used sex more of the time to help cope with stressful situations than did subjects who did not report unprotected anal intercourse. Unprotected anal intercourse was negatively associated with seeking social support and spiritual activities and positively associated with self-controlling coping, which involves keeping one's feelings to oneself, and positive reappraisal. The findings suggest that social aspects of coping may be a key to understanding differences between those who engage in high-risk sexual behavior and those who do not.


Stress, reactivity, and immune function in healthy men.

We examined the effects of acute psychological stress on lymphocyte proliferation and circulating levels of interleukin-1 and -2. Healthy men were exposed to two viewings of a gruesome surgery film and were asked to recall details of the film twice during a 30-min period. These subjects were compared to a nonstress control group. Lymphocyte proliferation to the mitogen concanavalin A (Con A; 5 micrograms/ml) was decreased during and after exposure to the stressor when compared to the control group. This decrease was more pronounced in subjects exhibiting greater blood pressure reactivity while viewing the film than in subjects showing smaller blood pressure responses. None of the other immunological measures was significantly affected by the stressor. Cortisol was not correlated with lymphocyte responsiveness. Possible explanations for these results and implications for further research are discussed.


Cognitive coping strategies and blood pressure responses to real-life stress in healthy young men.

Coping style is an important feature in the understanding of the relation between real-life stress and associated blood pressure (BP) responses. In this study, 10 high- and 10 low-"self-focused-coping" (SFC) male college students were tested with ambulatory BP monitoring on two typical schooldays, one of which included an examination. It was found that the high-SFC subjects, defined as those who tend to keep to themselves and/or blame themselves in stressful situations, showed higher BP responses than the low-SFC subjects, but only on the exam day. Further, the high-SFC subjects showed higher BP during the exam but also had BP elevations that were sustained during other activities throughout the same day, including evening rest. These results are discussed in terms of the relation between psychological and physiological responding.


Adult-child interaction during invasive medical procedures.

Adult-child interactions during stressful medical procedures were investigated in 43 pediatric patients videotaped during a venipuncture procedure in the course of cancer treatment. Relations among six adult behavior categories (explain, distract, command to engage in coping behavior, give control to the child, praise, and criticize/threat/bargain) and three child behavior categories (momentary distress, cry/scream, and cope) were examined using correlational and sequential analysis. Results indicated that adult distraction resulted in increased child coping and reduced momentary distress and crying. Adult explanations, although a likely response to child distress and crying, did not result in a reduction of these behaviors. Attempts to give the child control reduced child crying. Implications for clinical interventions during painful medical procedures are discussed.


Comparing the effectiveness of behavioral treatment for chemotherapy-induced nausea and vomiting when administered by oncologists, oncology nurses, and clinical psychologists.

Adequate control of side effects during medical treatment of cancer increases patient compliance and quality of life. Antiemetic drugs are not an effective treatment for the one in three cancer patients on chemotherapy who experience anticipatory nausea and vomiting (ANV); the behavioral treatment of systematic desensitization has been found effective for ANV when delivered by clinical psychologists. This study examined the effectiveness of systematic desensitization when delivered by medical personnel versus clinical psychologists. Seventy-two consecutive cancer patients with ANV were randomly assigned to no-treatment control or to systematic desensitization from 5 behaviorally trained clinical psychologists, 6 clinical oncologists, or 10 oncology nurses. The treatment was found effective in reducing anticipatory nausea, anticipatory vomiting, posttreatment nausea, and posttreatment vomiting compared to control patients, with no significant differences in effectiveness found between clinical psychologists and oncology staff. Although medical personnel should not engage patients in psychotherapy or other interventions that cannot be completed successfully, they can treat patients effectively with systematic desensitization and should be encouraged to learn and use this and other behavioral intervention techniques to benefit total patient care.


Assessing motivational readiness and decision making for exercise.

Motivational and cognitive processes of behavior change with respect to the area of exercise adoption were investigated. A total of 778 men and women, recruited from four worksites, answered a 40-item questionnaire consisting of statements based on constructs from the trans-theoretical model of behavior change. Principal-components analysis identified two factors--one a 6-item component representing avoidance of exercise (Cons), the other a 10-item component representing positive perceptions of exercise (Pros). Analysis of variance showed that the Pros, Cons, and a Decisional Balance measure (Pros minus Cons) were significantly associated with stage of exercise adoption. Results are consistent with applications of the model to smoking cessation and other areas of behavior change. Distinctions between exercise adoption and behaviors such as smoking cessation, weight loss, and alcoholism are discussed.


Trait humor and longevity: do comics have the last laugh?

Four sets of biographical data were analyzed in order to test the hypothesis that the ability to generate humor is associated with longevity. Although steps were taken to ensure that tests had high levels of statistical power, analyses provided very little support for the idea that individuals with a well-developed sense of humor live longer than serious writers and other entertainers. In addition, a subsidiary analysis revealed that those in the business of entertaining others died at an earlier age than those in other lines of endeavor. These findings suggest that researchers should turn their attention from trait humor to the effects of humorous material.


Culture and health-related schemas: a review and proposal for interdisciplinary integration.

We present a comprehensive review of anthropological, sociological, and psychological theory and data on the structure, content, and function of health-related schemas. Health psychology's need to integrate specific variables and principles from the other disciplines is highlighted. Suggestions for future research are offered, and the importance of cultural factors in health beliefs is emphasized.


National working conference on smoking and body weight. Task Force 1: Mechanisms relevant to the relations between cigarette smoking and body weight.

Careful, comprehensive, and empirical observations provide the building blocks of the sciences, whereas theory and mechanisms provide the "cement" to hold the blocks together and serve as blueprints to direct future building. This article resulted from several days of discussion regarding theories that may underlie the relation between cigarette smoking and body weight and the relation between smoking cessation and body weight. The working group composed of social and biological scientists who addressed this assignment considered what is already known within the smoking and body weight literature and also considered relevant findings from studies of smoking or body weight regulation that have not directly addressed the interaction of these variables. As expected, we were successful at listing some of what is not known and what is worth knowing. We also tried to identify fruitful possibilities for research activity that might clarify mechanisms of action and eventually lead to theoretical development. Because we do not believe that the present state of our deliberations merits the label of theories, we decided, instead, to report the summary of these deliberations as potential mechanisms relevant to the relation between smoking and body weight.


Plasma lipids, lipoproteins and post-heparin lipases in ponies with hyperlipaemia.

The metabolic origins of equine hyperlipaemia were investigated by analysing the concentration and composition of plasma lipoproteins in 18 ponies with the condition. The mean concentrations of cholesterol, triglyceride and very low density lipoproteins (VLDL) were increased by 4-, 52- and 19-fold, respectively, compared with a control group of 18 healthy ponies. These increases were due to the appearance of a buoyant VLDL fraction (VLDL1) not present in healthy ponies. The mean diameter of VLDL1 particles was 44% greater than control VLDL, and the particles were enriched in triglyceride and free cholesterol and depleted of cholesteryl esters, phospholipid and protein. The apolipoprotein (apo) B-100 content of VLDL1 was reduced and the ratio of apoB-100 to apoB-48 particles was 1:1, compared with 2:1 in control VLDL. The VLDL1 was also enriched in apoE, but had normal complements of apoC-II and apoC-III. The conventional VLDL (called VLDL2), LDL and HDL fractions were moderately enriched with triglyceride, and HDL contained increased amounts of apoE, apoC-II and apoC-III. The activities of lipoprotein lipase and hepatic lipase, the enzymes responsible for the catabolism of VLDL and their remnants, were increased by 2- and 3-fold, respectively, in response to the increased concentrations of their substrates. The composition of VLDL1 suggested that the liver was maximising the secretion of triglyceride by producing larger number of VLDL particles that accommodated a greater mass of triglyceride by having apoB-48 rather than apoB-100 as their structural protein. Plasma free fatty acid (FFA) concentrations were elevated in 17 of the 18 ponies, suggesting that increased FFA flux might be the stimulus for hepatic triglyceride synthesis and VLDL secretion. We conclude that overproduction, rather than defective catabolism, of VLDL was the cause of the hyperlipidaemia and that lipid lowering agents which reduce VLDL synthesis, by decreasing adipose lipolysis and FFA flux, are candidates for the management of hyperlipaemia.


Five cases of gastrocnemius tendinitis in the horse.

The normal gastrocnemius tendon may contain some muscular tissue proximally. This results in a patchy echogenicity ultrasonographically where it lies caudal or lateral to the superficial digital flexor tendon (SDFT). When it has assumed a position dorsal (cranial) to the SDFT the gastrocnemius tendon has a more uniform echogenicity and its margins are well defined. Five horses had lameness associated with lesions identified ultrasonographically in the gastrocnemius tendon in the latter region. Lameness ranged from mild to severe and was characterised by reduced hock flexion, lowered arc of foot flight, shortened length of the cranial phase of the stride and in some horses a reduced duration of weight bearing during the caudal phase of the stride. Lameness was variably accentuated by flexion of the proximal or distal limb joints of the lame limb. Perineural analgesia of the tibial and fibular nerves or the tibial nerve alone substantially improved the lameness. All horses remained lame 2-3 months after initial examination, with minimal change in the ultrasonographic appearance of the lesion(s).


Retrospective study of 38 cases of femur fractures in horses less than one year of age.

Medical records of 38 horses less than 1 year of age and diagnosed as having a fracture of the femoral diaphysis, metaphysis or distal physis were evaluated. Twenty-six foals had fractures of the femoral diaphysis or metaphysis with the most common fracture configuration being comminuted. Twelve foals had distal physeal fractures with the most common fracture configuration being a Salter-Harris type II. Twenty-one foals with fractures of the capital femoral physis, neck or greater trochanter during the same time period were excluded from this study. Surgical repair was attempted in 16 diaphyseal and 2 distal physeal fractures. Most of the diaphyseal fractures were repaired by placing plates on the lateral and cranial surfaces of the bone. Dynamic condylar screw plates or angle blade plates were used for increased bone purchase in 4 foals with short distal fragments. Five foals with distal physeal fractures were treated; 2 were surgically treated by placing an angle blade plate on the lateral cortex, and 3 foals with minimally displaced distal physeal fractures were managed with stall confinement. Eight of the 16 surgically repaired diaphyseal fractures healed. Fracture location and configuration was not a determinant of outcome, but the mean age of foals with successfully repaired diaphyseal fractures was 2 months compared with 4 months for the unsuccessful cases, indicating that the age and size of the foal was important. Long-term follow up revealed that 6 of the 8 successfully repaired diaphyseal fractures had no residual effects of the fracture observed during performance of the horse for its intended use. Only 1 of the 2 surgically repaired distal physeal fractures healed, but this horse was eventually killed because of unthriftiness related to a malabsorption syndrome. Some form of complication developed in 13 of the 18 surgically repaired fractures. Infection was the primary cause of failure. The greatest determinant associated with infection was the inability to control post-surgical seroma formation.


Tearing of the medial palmar intercarpal ligament in the equine midcarpal joint.

Tearing of the medial palmar intercarpal ligament is described in 45 intercarpal (midcarpal) joints in 42 horses (37 racehorses, 5 non-racehorses). Of the 37 racehorses, there were 20 Quarter Horses, 14 Thoroughbreds and 3 Standardbreds. The patients had been referred for arthroscopic surgery for removal of osteochondral chip fragments that had been diagnosed radiographically or diagnostic arthroscopy of a persistent carpal problem. The problem was unilateral in 39 horses and bilateral in 3. The presenting clinical signs were lameness and/or persistent synovial effusion. In one instance, the presenting complaint was haemarthrosis. Osteochondral chip fragments were present in the joint affected with tearing in 23 horses. In 6 horses in which osteochondral fragments were present in other joints, the degree of synovial effusion was greatest in the midcarpal joint with ligamentous tearing. In most of the 22 midcarpal joints where carpal chip fragmentation and ligamentous tearing were present concomitantly, the degree of clinical compromise was greater than normally seen with that degree of osteochondral fragmentation. A ligament was designated as torn when a defect was present in the ligament. This usually took the form of frayed fibres suspended in the irrigating solution, presenting a transverse type of defect in the dorsal aspect of the lateral portion of ligament. However, longitudinal tearing was present in 1 case and tearing was noted in the palmar aspect of the ligament in 2 other cases. The shredded fibres were trimmed in most cases and this allowed better definition of the amount of ligament considered to be torn. The degree of damage ranged from 10% to 100% of the width considered to be torn.(ABSTRACT TRUNCATED AT 250 WORDS)


Idiopathic muscular hypertrophy of the equine small intestine: 11 cases (1980-1991).

The medical records of 11 horses with idiopathic muscular hypertrophy (MH) of the small intestine were reviewed to determine the clinical and pathological features of the disease. The median age of affected horses was 10.0 years (range 5-18 years). No breed or sex predisposition was apparent. Ten horses (91%) had chronic (23 days to 2.4 years) signs of mild, intermittent colic, and 1 horse had signs of severe colic of only 3 days' duration. Partial anorexia and chronic weight loss of variable duration (1-6 months) were prominent historical findings in 5 (45%) horses. Diagnostic tests, with the exception of exploratory caeliotomy, were ineffective for definitive diagnosis of intestinal MH as a cause of colic. In 2 horses, however, a thickened, rigid ileum was detected by palpation per rectum, and in 5 horses, multiple loops of distended small intestine were detected by palpation per rectum. Hypertrophy of both the circular and longitudinal layers of muscularis was determined as the cause of intestinal thickening in all horses. Muscular hypertrophy of the ileum was present in 9 (82%) horses. Two horses (18%) had MH of a section of jejunum only, and 4 (36%) horses had MH of the ileum in combination with MH of other sections of small intestine. Two (18%) horses had MH of the entire small intestine. In 9 (82%) horses, intestinal MH resulted in narrowing of the luminal diameter at the site of MH. Small diverticula were present on the mesenteric border of the hypertrophied ileum of 5 (45%) horses. Five linear (up to 150-cm) diverticula were present in the hypertrophied jejunum of 1 (9%) horse. Haemomelasma ilei was present on the antimesenteric serosal surface of affected intestine of 8 (73%) horses. Full-thickness rupture of the ileum with subsequent diffuse, septic peritonitis occurred in 3 (27%) horses.


Arachidonate metabolites in bronchoalveolar lavage fluid from horses with and without COPD.

Arachidonate metabolites were measured in bronchoalveolar lavage fluid (BALF) from horses with (N = 4) and without (N = 7) chronic obstructive pulmonary disease (COPD). Prostaglandin (PG) D2, leukotriene (LT) B4 and LTC4 were present in highest concentrations in BALF from clinically normal horses. Concentrations of PGE2 and PGF were significantly higher in BALF from horses with COPD than in BALF from normal horses, but no differences were detected in thromboxane B2, 6-keto-PGF1 alpha, PGD2, LTB4 or LTC4.


Healing of full-thickness cartilage compared with full-thickness cartilage and subchondral bone defects in the equine third carpal bone.

The effect of lesion depth on the quality of third carpal bone cartilage repair was examined. A 1-cm diameter articular defect penetrating the calcified cartilage in one limb and the subchondral bone plate in the opposite limb was created in the radial facet of the third carpal bones. Clinical and xeroradiographic examinations were performed every 4 weeks until 4 months (3 horses) and 6 months (3 horses) after surgery. The synovial membrane, non-opposing articular surfaces and articular defects were examined grossly, histologically and histochemically. Grossly, deeper defects contained thicker, whiter tissue, but both joints contained generalised degenerative changes. Defects extending through calcified cartilage were filled deeply by fibrocartilage and superficially by fibrous connective tissue. Defects extending through subchondral bone were consistently filled with hyaline-like cartilage in the depths of the lesion, fibrocartilage in the intermediate layer and fibrous connective tissue superficially. The results indicate that subchondral bone is the source of hyaline-like cartilage repair tissue and suggest that quality of healing of cartilage defects may be improved by penetrating the subchondral bone plate. It also appears that the synovitis associated with the procedure must be controlled before the procedure can be advocated for treatment of clinical cases.


Comparison of two grafting methods in 4.0-mm drill defects in the third metacarpal bone of horses.

In 6 horses, bilateral metacarpal vertical series of three 4.0-mm unicortical drill holes were made. At random, one of each series of 3 holes was filled using a sternal 4.0-mm cancellous bone cylinder or a slurry of cancellous bone injected into the hole or left as an empty control. All horses had lateral metacarpal xeroradiographs at monthly intervals. Three horses (6 metacarpi) were examined post mortem after 4 months and 3 others after 6 months. Immediate through 4-month post-operative xeroradiographs demonstrated increased density in the holes with cancellous cylinders and no difference could be seen between the untreated controls and holes injected with slurry. From 5 months, no radiographic difference could be seen between the treatment groups. No consistent histological difference between treatment groups could be detected. In conclusion, no justification for clinical grafting of 4.0-mm unicortical dorsal metacarpal drill holes could be found.


Intravenous catheterisation of foetus and mare in late pregnancy: management and respiratory, circulatory and metabolic effects.

The uterine and umbilical vessels of 12 pregnant ponies were catheterised to study foetal metabolism. The effects of this procedure on maternal and foetal cardiovascular, respiratory, metabolic and adrenocortical activity were monitored during and after surgery. Premedication with acepromazine-butorphanol-detomidine was followed by induction of anaesthesia with detomidine and ketamine and maintenance, using mechanical ventilation, with halothane in oxygen and nitrous oxide. Mean maternal arterial blood pressure was greater than 70 mmHg during anaesthesia and arterial oxygen tension remained over 100 mmHg. The foetuses were adequately oxygenated but were hypercapnic and lactic acidaemic. Most maternal and foetal blood gases and metabolites had returned to normal by 24 h, although foetal plasma lactate fell more slowly. The maternal adrenocortical discharge was less severe than reported previously and plasma cortisol had fallen to basal levels by 48 h after surgery. Foetal plasma cortisol remained low and did not change during or after surgery. Arterio-venous metabolite and gas tension differences across the uterine and umbilical circulations were slightly greater at operation than in the recovery period, suggesting that uteroplacental perfusion may have been impaired during surgery. Post-operative recovery of the mare and foetus was satisfactory and subsequent problems associated with the foetal catheters were not related to the anaesthesia or surgery.


Production of mitogen-contamination free alginates with variable ratios of mannuronic acid to guluronic acid by free flow electrophoresis.

Commercial alginates consisting of variable homopolymeric regions of beta-D-mannuronic acid and alpha-L-guluronic acid, interspaced with regions of alternating blocks, are potent stimulators of macrophages and lymphocytes. Therefore, inflammatory reactions and fibrotic overgrowth of the beads result if Langerhans islets are encapsulated in raw alginate hydrogel beads (cross-linked with divalent cations). The result is random failure of the islets some time after transplantation. Analysis of raw alginates by using free flow electrophoresis demonstrated that commercial alginates contained at least 10-20 fractions (characterized by different electrophoretic mobilities) which showed mitogenic activity. These fractions could be quantitatively separated from the alginic acids by free flow electrophoresis on a preparative scale. The purified alginates cross-linked with Ca2+ ions exhibited no mitogenic reactions as proved by an in vitro assay. In addition, examination of purified Ba2+ alginate beads implanted intraperitoneally in rats or mice for three weeks showed no fibrotic overgrowth in contrast to implants made from unpurified alginate.


Steady-state heat transfer and thermal zone spreading in gel isoelectric focusing.

The zone spreading caused by a transverse pH profile due to a temperature gradient through the thickness of a gel slab is estimated. The temperature difference (delta T) between the upper and lower gel surfaces can be calculated as a function of the electric power dissipated in the gel and the gel dimensions. It is found that when delta T is only 1 degree C the zone spreading due to this thermal excursion is as high as 0.5 mm. Thus, an admissible delta T is found to be equal to 0.2 degrees C, since this corresponds to a thermal zone spreading of only 0.1 mm, i.e. the same order of magnitude as the spatial resolution of a laser scanner. A thermal zone spreading of 0.1 mm is compatible with a resolving power of 0.01 pH unit, the current limit of conventional isoelectric focusing in amphoteric buffers. A requirement for the thickness of a gel slab is formulated: e.g., at 40 W applied power (over a gel surface area of 25 x 11 cm), a thermal zone spreading limited to 0.1 mm can only be obtained with a gel thickness of approximately 170 microns.


Temperature gradient gel electrophoresis: rapid detection of alpha-1-antitrypsin deficiency carriers.

The homozygous state of the alpha-1-antitrypsin (alpha 1AT) deficiency variant Z is associated with severe liver damage in early childhood and progressive lung emphysema in adulthood. A single base transition (G to A in codon 342) in exon V is causing the severe disease. The Glu342 to Lys342 mutation can be detected conventionally by isoelectric focusing (IEF) or on the DNA level by the newly developed method of temperature gradient gel electrophoresis (TGGE). It is the aim of this study to describe the TGGE technique, to compare the results with conventional IEF, and to discuss its efficiency for different diagnostic applications.


Concave Ferguson plots of DNA fragments and convex Ferguson plots of bacteriophages: evaluation of molecular and fiber properties, using desktop computers.

A desktop computer program evaluating physical properties of DNA and bacteriophages is presented. The analysis is based on data obtained from capillary and submarine-type agarose electrophoresis. Native molecular/particle properties and properties of the gel (or polymer) medium can be derived from electrophoresis at several gel concentrations. This is done conveniently by a computerized evaluation of the semi-logarithmic plot of mobility vs. gel concentration, designated the Ferguson plot. In application to most proteins, this plot is linear and computer programs exist to evaluate it. However, nonlinear Ferguson plots have assumed great importance in view of the fact that the plots are concave for DNA. Similarly, convex plots are important since they prevail in the electrophoresis of large particles in agarose. The computer program reported here is the first to (i) address concave Ferguson plots and (ii) allow for the evaluation of both cases using a desktop computer. Program ELPHOFIT version 2.0, a Macintosh application, is available upon request.


Increased electrophoretic mobility of sodium sulfite-treated jack bean urease.

Sodium sulfite is a widely used activity-protective agent for the storage of urease. However, this reagent produces a 10% increase in the anodic electrophoretic mobility of native urease. Changes in the hydrodynamic properties of the enzyme are not involved in that modification. The observed change is related to an increased negative charge of the protein molecule in the presence of sodium sulfite. The results are discussed in terms of sulfitolysis of the single disulfide bond in the urease monomer. It is remarkable that the modification occurs at neutral pH. Our results show that removing sodium sulfite and reversing its effect by treatment with 2-mercaptoethanol are required prior to any study involving native urease.


Two-dimensional electrophoretic analysis of transformation-sensitive polypeptides during chemically, spontaneously, and oncogene-induced transformation of rat liver epithelial cells.

Recently, we described the establishment of a computerized database of rat liver epithelial (RLE) cellular polypeptides (Wirth et al., Electrophoresis, 1991, 12, 931-954). This database has now been expanded to include the analysis of cellular polypeptide alterations during chemically (aflatoxin B1; AFB), spontaneously, and oncogene (v-Ha-ras, v-raf, and v-myc/v-raf)-induced transformation of RLE cells. Two-dimensional mapping of [35S]methionine-labeled whole cell lysate, cell-free in vitro translation products and [32P]orthophosphate-labeled polypeptides revealed subsets of polypeptides specific for each transformation modality. A search of the RLE protein database indicated the specific subcellular location for the majority of these transformation-sensitive proteins. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin- and intermediate filament-related polypeptides (vimentin, beta-tubulin, the cytokeratins, and actin) were observed among the various transformant cell lines. Immunoprecipitation and Western immunoblot analysis of tropomyosin expression in four individual AFB-, as well as four spontaneously induced, and each of the oncogene-transformed cell lines indicated that five major tropomyosin (Tm 1-5) isoforms were variably expressed in the various cell lines, including one polypeptide tentatively identified as Tm6. Whereas alterations in tropomyosin expression appeared to be transformation-specific, alterations in the individual intermediate filament polypeptides were related more to the differentiation state of the individual cell lines rather than to the transformation phenotype. These studies extend our earlier efforts toward the establishment of a comprehensive computerized database of RLE cellular proteins and demonstrates how such a database may serve as a useful source for studies concerning the regulation of growth and differentiation as well as transformation of RLE cells.


High resolution two-dimensional gel analysis of proteins in the central nervous system of larvae of Drosophila melanogaster.

An improved method of high resolution two-dimensional gel electrophoresis was used to study the patterns of protein synthesis in the central nervous system (CNS) of late instar larvae of Drosophila melanogaster. A small number of CNSs was radiolabeled with a mixture of 14C-labeled amino acids or with [35S]methionine, and the pattern of labeled proteins was analyzed. One thousand and forty-five polypeptides, 800 acidic (IEF) and 245 basic (NEPHGE), from the CNS of several wild-type strains have so far been separated and cataloged. All these polypeptides were numbered and compared with our catalog of polypeptides from wing imaginal discs, and quantitative or qualitative changes were detected in more than 23% of the polypeptides. When comparing patterns of label of CNSs we found small quantitative differences in the rate of synthesis between individuals of the same strain, not due to sexual differences, and a minute number of quantitative and qualitative differences between groups of individuals of different strains.


Two-dimensional polyacrylamide gel electrophoresis reveals differences between osteoblast and fibroblast extracellular proteins.

Normal human skin fibroblast primary cell lines secrete over 50 proteins into culture medium. These have been mapped previously using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and this technique has now been used to investigate extracellular protein secretion by human osteoblasts in vitro. We report the mapping of a number of consistent markers specific to the osteoblast. In particular, one protein chain with posttranslational modifications was found to be unique to the osteoblast extracellular protein map. The absence of the N- and O-glycoforms of collagenase from the osteoblast profile in this study concurs with findings reported using the immunoprecipitation functional assay and Northern blot analysis. The use of 2-D PAGE in phenotypic assessment provides a more complete analysis than the standard range of single-parameter tests for osteoblasts. Mapping of extracellular and cellular proteins in addition to bone matrix protein analysis will allow a comprehensive analysis of normal osteoblast function. This technique may also be applied to the study of osteoblasts in relation to bone disease and in assessing the phenotypic shift within a normal osteoblast culture.


DNA-protein interaction analysis using polyacrylamide gel electrophoresis and a simple and sensitive UV crosslinking procedure.

A simple and reproducible technique for DNA-protein interaction analysis is described using UV crosslinking and polyacrylamide gel electrophoresis, leading to visualization of the complexes as distinct and strong signals. It avoids incorporation of bromodeoxyuridine (BrdU) into DNA and requires no special equipment. It was successfully applied to an oligonucleotide sequence from within the first intron of the mouse myb proto-oncogene and nuclear extracts from a myb-expressing cell line.


An experimental study of the effect of isoflurane on epileptiform bursts.

The effect of isoflurane on penicillin- and picrotoxin-induced epileptiform activity was tested using hippocampal slice preparations. Isoflurane reduced both the frequency of spontaneous epileptiform bursts and the number of population spikes within each burst in a dose-dependent manner. The last population spikes in the burst were most sensitive to the anesthetic, whereas the first 4-6 spikes were quite resistant and persisted until spontaneous activity was abolished at 3% isoflurane. Isoflurane increased the stimulus current required to evoke epileptiform bursts and shifted the relationship between stimulus current and population spike amplitude to the right. At 3% isoflurane, a dose that usually causes iso-electric EEG and abolishes all spontaneous epileptiform activity, responses could still be evoked, and then invariably had an epileptiform pattern. The maximum response was reduced compared to control and 1.5% isoflurane. With isoflurane there was a reduced tendency for activity to be transmitted from one region within the hippocampus to the other. This effect was also dose-dependent. However, transmitted activity always retained a typical epileptiform character, although the number of population spikes within a train to some extent decreased with increasing concentrations of isoflurane.


Positive transfer of audiogenic kindling to electrical hippocampal kindling in rats.

Audiogenic seizures in genetically susceptible rodents are provoked by intense acoustic stimulations which result in a tonic seizure associated with a short flattening of the EEG. These seizures have been shown to involve primarily brainstem structures. Daily exposure to sound for 30-40 days produced a permanent change in the evoked seizure with development of facial myoclonias, rearing and falling, or of tonic-clonic seizures accompanied by high amplitude cortical spike-and-wave discharges. Kindled audiogenic seizures appear similar to seizures kindled from amygdala or hippocampus, suggesting that repeated auditory stimulations cause a progressive propagation of the epileptic discharge toward limbic structures. To verify this hypothesis, the behavioral and EEG development of electrical hippocampal kindling has been studied in 7 non epileptic controls (NE), 8 acoustic susceptible (AS), and 8 audiogenic kindled rats (KAS). The behavioral and EEG development of the electrical hippocampal kindling was similar in the AS and the NE rats. However, 2 animals in the AS group but no controls exhibited behavioral running and bouncing during the course of hippocampal kindling. In the KAS group, the hippocampal kindling was clearly facilitated as compared to NE and AS: behavioral stage greater than or equal to 5 was reached in a mean of 4 stimulations in KAS versus 30 and 22 stimulations respectively in NE and AS groups. This positive transfer phenomenon suggests that during kindling of audiogenic seizures, epileptic discharge spreads from the brainstem to the forebrain and progressively involves the hippocampus.


Effects of 3-hydroxy,3-ethyl,3-phenylpropionamide (HEPP) on rat models of generalized and focal epilepsy.

The GABA withdrawal syndrome (GWS) is a new model of focal epilepsy in which paroxysmal activity is induced through the interruption of a chronic, intracortical infusion of GABA. Preliminary studies have shown extraordinary resistance of this epileptogenic activity to classic anticonvulsants including diazepam, the most effective agent for treating status epilepticus. However, GWS can be inhibited by GABA itself. The rat with petit mal-like seizures is a genetic model of generalized non-convulsive epilepsy (GNCE), with behavioral characteristics and electrical (spike-and-wave discharges) signs resembling absences. Moreover, GABAmimetics aggravate this type of seizure. Rats with GWS induced by cessation of a localized GABA infusion (50 micrograms/microliters/h for 24 h), and the rat model of GNCE, were treated with HEPP, a new anticonvulsant agent. In the case of GWS, the drug produced a significant decrease of focal spike activity in animals which started discharging at low frequencies while in rats with higher frequency discharge, HEPP was without effect. HEPP administered on the second day of the GWS in naive rats had no effect. In rats with GNCE, doses of 50 and 100 mg/kg i.p. blocked the spike-and-wave discharges. The higher dose produced sedation in this absence seizures model. Although the mechanism of action of HEPP is still unknown, its unique antiepileptic profile deserves further studies.


Section of the corpus callosum in kainic acid induced seizures in rats: behavioral, electroencephalographic and neuropathological study.

Clinical and experimental data suggest that the role of corpus callosum in epilepsy includes synchronization, spread, excitation and inhibition. Section of the corpus callosum (SCC) is known to be a useful therapy in selected types of generalized epilepsy, i.e., tonic, atonic and generalized convulsive seizures, but not partial seizures which may be exacerbated by this procedure. The goal of this study was to determine the effect of SCC in the kainic acid (KA) model of limbic seizures in rats. Using several doses of KA (2.5, 5 and 10 mg/kg) injected systemically, we found a potentiation of the behavioral, electrographic and histological effects of KA in the SCC group of animals compared to the sham-operated control rats. A low dose of kainic acid (2.5 and 5 mg/kg) induced status epilepticus in the SCC animals, but not in the sham-operated control rats. These data demonstrate that in the KA model of temporal lobe seizures, SCC not only fails to protect, but actually intensifies seizures. This finding is compatible with the hypothesis that there is an inhibitory influence, via the corpus callosum, of the non epileptic neocortex on its contralateral homologue in the kainic acid model.


Corpus callosotomy in the lithium-pilocarpine model of seizures and status epilepticus.

Section of the corpus callosum (SCC) is a useful surgical therapy in selected types of epilepsy, i.e., tonic, atonic, and intractable generalized convulsive seizures. Experimentally, the effects of SCC have been documented in animal models of focal seizures as well as generalized seizures. The object of this study was to determine the effect of SCC on behavioral and EEG symptomatology in the lithium-pilocarpine model of seizures and status epilepticus in the rat. SCC was well tolerated. Fifty-seven percent of SCC animals never developed status epilepticus, while all control animals developed status epilepticus. None of the SCC animals died after 24 h but 59% of control animals died within 24 h of status. Histology verified the extent of the SCC and demonstrated widespread brain damage in all animals who exhibited status epilepticus after 72 h. SCC was associated with a lesion of hippocampal commissure in 64% of animals in the SCC group. This protective effect was not related to lesion of the skull or the longitudinal sinus. The lesion of the hippocampal commissure may have contributed to the protective effect of SCC, since animals with an isolated lesion of the hippocampal commissure without SCC survived the status and showed an increased latency to seizure and status epilepticus. These data suggest that the lithium-pilocarpine model of status epilepticus may be useful in the study of the mechanism of efficacy of SCC in the treatment of epilepsy.


Variability and clinical relevance of the interaction between sodium valproate and carbamazepine in epileptic patients.

Twenty-four epileptic patients (16 females, 8 males; aged 13-62 years) were studied before and after the addition of sodium valproate (VPA) 500 mg twice daily for 5 days. All had been established previously on carbamazepine (CBZ) as monotherapy (300-1600 mg daily in divided doses). Sixteen of these patients undertook a battery of cognitive function tests before and after VPA introduction. VPA had no effect on total or free CBZ concentrations. However, median concentrations of the active metabolite, CBZ 10,11 epoxide (CBZ-E), were significantly increased (CBZ-E before VPA 1.3 mg/l, after VPA 2.1 mg/l, P less than 0.01). The median rise was 25%, although the extent of the interaction ranged from a 25% decrease to an increase of 123% in CBZ-E concentrations. This was related to the marked inter-individual variation in circulating VPA (mean 25-69 mg/l), as CBZ-E concentrations correlated significantly with total (r = 0.5, P less than 0.05, 95% CI 0 to +0.08) and free (r = 0.7, P less than 0.001, 95% CI +0.09 to +0.25) VPA levels in individual patients. Although uncontrolled, no deterioration in performance of any of the cognitive function tests was observed following the addition of VPA. This study does not support immediate clinical relevance for this drug interaction between VPA and CBZ.


Cognitive effects of oxcarbazepine and phenytoin monotherapy in newly diagnosed epilepsy: one year follow-up.

We evaluated the effect of initial oxcarbazepine (OXC) monotherapy on memory, attention and simple psychomotor speed in 14 patients; 15 patients with initial phenytoin (PHT) monotherapy served as reference patients. Neuropsychological assessments were performed before starting the treatment and after 6 and 12 months follow-up with steady-state drug treatment. Differential cognitive effects of OXC and PHT were not apparent in our study. As the efficacy of present antiepileptic drugs in adult epilepsy is analogous, the choice of drug is determined by the comparative side effects of the drugs. In the present study the number of successfully treated patients was similar in both OXC and PHT groups. As far as cognitive side effects are concerned our results revealed no evidence favoring either antiepileptic over the other.


Help-seeking strategies for epilepsy by previously untreated patients in northern Ecuador.

Two hundred and fifteen patients with epilepsy and 125 controls were given questions designed to elicit their help-seeking strategies for the condition. This was part of a large-scale medical intervention study in a rural area of a developing country (the provinces of El Carchi and Imbaburra, northern Ecuador). Local medical personnel were also surveyed. One hundred and forty-four patients and 98 controls were questioned again 12 months after they had been enrolled in the intervention programme to see if their attitudes and practices had changed. Over the study period, belief in medical remedies, particularly those offered by the study, rose significantly among both controls and patients. Control readiness to consult local healers for help and belief in their effectiveness also rose, whereas patient belief in healer help, already significantly less at baseline because of previous negative experiences, had declined still further. Though no patients treated were on antiepileptic treatment at baseline, 71% claimed to have sought the help of allopathic practitioners at some point in the past, 35% 'many times'. Of these only 21% had ever been given antiepileptic drugs; some consultations were not at local health facilities. Thirty-two per cent of patients also claimed to have consulted traditional healers. The high number of patients claiming to visit doctors was not entirely confirmed in the medical evidence. Even this source, however, suggested more consulted doctors than expected from the observation that only 10% were on any kind of treatment at the start of the study (none on antiepileptic drugs).(ABSTRACT TRUNCATED AT 250 WORDS)


Properties of the convulsive threshold determined by direct cortical stimulation in rats.

The threshold for convulsions in rats can be determined by applying ramp-shaped pulse trains directly to the cerebral cortex in rats, which provides a convenient model for investigating anticonvulsant drug effects. This study was undertaken to extend a previous study on the properties of this model. Analysis of the cortical EEG, recorded from two motor areas and one somatosensory area, showed that the start of clonic forepaw movement, marking the convulsive threshold, is preceded by the appearance of sharp negative spikes at the electrodes in the two motor areas. There was a strong linear relation between the clinically determined threshold and the EEG derived threshold (r = 0.93, slope 0.99, SD 0.04), confirming the validity of the clonic movement threshold as an objective and accurate measure. Examination of the seizure patterns seen with various degrees of suprathreshold stimulation led to the distinction between a threshold for localized and for generalized seizure activity (TLS and TGS respectively). Carbamazepine selectively and strongly increased the TGS, whereas it only slightly affected the TLS, indicating that cortical stimulation can be used to select drugs that specifically prevent seizure spread, for which carbamazepine is a prototype. It was found that the TLS was not affected by testing at intervals as short as 1 min, provided that no self-sustained seizures were induced. However, if the TGS was passed, the TLS was increased substantially for at least 10 min, while complete recovery could take several hours. The intensity of stimulation, rather than seizure duration, appeared to be the determinant for the TLS increase. There was no seasonal influence or effect of stimulation electrode depth. There may be a minor effect of experience in using the test. It was concluded that the observed variability was mainly an intrinsic property of the individual animal.


Regional brain glucose metabolism in patients with complex partial seizures investigated by intracranial EEG.

We performed interictal 18F-2-fluoro-2-deoxy-D-glucose positron emission tomography (18FDG-PET) studies in 57 patients with complex partial epilepsy (CPE), not controlled by medical treatment and considered for surgical resection of their epileptic focus. A precise localization of the epileptic focus was obtained in 37 of these patients with a combination of subdural and depth electrodes. We visually inspected the metabolic images; we also measured glucose consumption in a number of brain regions and compared the values with those obtained in 17 normal controls. Eighty-two percent of the 57 patients had an area of glucose hypometabolism on the 18FDG-PET images. Six patients had a frontal epileptic focus, 3 of them had a frontal lobe hypometabolism. Twenty-six patients had a unilateral temporal lobe focus and all of them displayed a temporal lobe hypometabolism. The asymmetry was more pronounced in the lateral temporal cortex (-20%) than in the mesial part of the temporal lobe (-9.6%). In each cortical brain region on the side of the epileptic focus (except the sensorimotor cortex), glucose consumption rate was lower than in the contralateral region or than in controls. No differences could be found between patients with a seizure onset restricted to the hippocampus and patients with a seizure onset involving the hippocampus and the adjacent neocortex. Divergent metabolic patterns were obtained in 5 patients with bilateral temporal seizure foci. Combined with other non invasive techniques (EEG, neuroradiology), PET contributes increasingly to the selection of patients with CPE who could benefit from surgical treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Single photon emission computed tomography using perfusion tracers in seizure disorders.

Single photon emission computed tomography (SPECT) using perfusion tracers makes it possible to estimate regional cerebral blood flow (rCBF) and, indirectly, local brain metabolism. It may be possible to detect and follow physiopathological alterations, such as may be seen in seizure disorders. The authors review the principles of and some data on perfusion SPECT in seizure disorders, stress advantages as well as major drawbacks and add their initial experience with Tc-99m hexamethylpropyleneamine oxime (HMPAO) SPECT in febrile convulsions.


Polyamine metabolism in epileptic cortex.

Polyamine (tissue) concentrations have been studied in hippocampus and temporal neocortex from patients with temporal lobe epilepsy. Depth electrode recordings demonstrated hippocampal origin of the seizures, the temporal neocortex being involved during the discharge propagation. Neuropathological examination of excised tissues showed glial proliferation or glioma in Ammon's horn (CA), whereas the temporal neocortex did not exhibit any histological abnormality. Polyamine (putrescine or PUT, spermidine or SPD, spermine or SPM) concentrations were determined on surgical samples from the hippocampus and various areas of temporal neocortex. Human post-mortem tissue from temporal lobe regions was used for controls. In post-mortem controls and temporal neocortex specimens from epileptic patients, polyamine levels were similar (in nmol/g wet weight: PUT = 40-100; SPD = 200-350; SPM = 100-200). In CA, polyamine levels exhibited striking changes: SPD content was significantly increased (350-700 nmol/g) while SPM was lowered (50-100). PUT was only increased in CA invaded by the tumoral process (100-180). Accordingly, a very high SPD/SPM molar ratio in the abnormal CA region was observed, indicating an acceleration of polyamine neosynthesis which is usually related to ornithine decarboxylase induction. Metabolic changes in polyamines appear to be selective of human epileptic hippocampus. A relationship between glial proliferation (gliosis or neoplasia), epileptic firing and polyamines is discussed.


Aspects of pharmacotherapy in epilepsy.

Notwithstanding important advances in the treatment of epilepsy basic knowledge about the epilepsies and about the mechanism of action of antiepileptic drugs is still fragmentary. This statement is illustrated with examples from the laboratory and clinical practice. In particular it is emphasized that not only is little known about the mechanism of side-effects but also little work appears to be in progress to change that situation.


A brief history of epilepsy and its therapy in the Western Hemisphere.

The history of epilepsy and its treatment in the western world dates back at least 4 millennia to the ancient civilization of the middle east. Past and present treatments have been empirical, usually reflecting the prevailing views of epilepsy, be they medical, theological or superstitious. Ancient physicians relied on clinical observation to distinguish between epileptic syndromes and infer their cause. Early pathophysiological theories of epilepsy correctly identified the brain as the site of the problem, but emphasized incorrect causes such as an excess of phlegm in the brain. Treatments consisted of prescribed diets or living conditions, occasional surgery such as bloodletting or skull trephination and medicinal herbs. These treatments, often ineffective, had the intellectual advantage of being based on pathophysiological principles, unlike current, more empirical, therapies. The unfortunate but widely held view of epilepsy as being due to occult or evil influences gained adherents even in the medical world during ancient times, and the later acceptance of Christianity allowed theological interpretations of seizures as well. Magical or religious treatments were more frequently prescribed as a result, practices which persist to this day. In the Renaissance an attempt was made to view epilepsy as a manifestation of physical illness rather than a moral or occult affliction, but it was during the Enlightenment that epilepsy was viewed along more modern lines, helped by advances in anatomy and pathology and the development of chemistry, pharmacy and physiology. The idea that focal irritation may cause seizures came about from clinical and experimental work, and was supported by the successful control of seizures by the (sedative) bromides and barbiturates in the late 19th century. The introduction of phenytoin showed that non-sedative drugs could be effective in controlling seizures as well, and the development of in vivo seizure models widened the scope of pharmaceutical agents tested for their efficacy against epilepsy. Increasing knowledge of the cellular mechanisms of epilepsy will, hopefully, allow the development and introduction of drugs with increasing specificity against seizure activity and the development of epilepsy.


Genetic models of absence epilepsy, with emphasis on the WAG/Rij strain of rats.

In this review, the main characteristics of genetic models of absence epilepsy, in particular with respect to WAG/Rij rats, are presented. Genetic models are important and relevant, since evidence exists that these models mimic spontaneously occurring human epilepsy more than models in which epilepsy is artificially induced. Genetic models can be divided into models in which seizures are elicited and into those in which epilepsy appears without any sensory stimulation. The majority of genetic models show that absence type of epilepsy; during the last few years, we and others have noticed that rats of various strains exhibit spontaneously occurring spike-wave discharges in the EEG. Among the strains highly affected is the WAG/Rij strain, which is a fully inbred strain. Individuals are homozygous and because of this property, genetic studies are meaningful. Electrophysiological studies have indicated that abnormal discharges in the cortical EEG are generalized and that the hippocampus is not involved. Parts of the thalamus, together with the thalamic reticular nucleus, apparently act as a pacemaker for the abnormal discharges. There is a circadian modulation in the number of spike-wave discharges. Discharges mainly occur during intermediate levels of vigilance such as passive wakefulness and light slow-wave sleep and at transitions of sleep states. Pharmacological studies with clinically effective antiepileptic drugs have shown a close agreement in seizure response between man and rat. Studies with new compounds have emphasized the role of the GABAergic and glutamatergic system in this type of epilepsy. Particularly striking is the role of the GABAergic system. GABA agonists enhance and GABA antagonists reduce the occurrence of spike-wave discharges, which deviates from the effects of GABAergic drugs in non-convulsive epilepsy. Even more striking is the role of the benzodiazepines, generally seen as GABA agonists; these drugs do not act as such in absence epilepsy since they reduce spike-wave discharges. Also good evidence for an involvement of other neurotransmitters such as noradrenaline, dopamine and opioid peptides exists in absence epilepsy. Genetic data obtained from the WAG/Rij model for absence epilepsy show a relatively simple pattern of inheritance with one gene determining whether an individual is epileptic or not, and with other genes regulating the number and duration of seizures. This is in good agreement with the more restricted human data. Cognitive studies have shown two important features of epilepsy in the WAG/Rij strain: modulation of the number of spike-wave discharges by mental or physical activity and on the other hand, the disruption of cognitive activity by spike-wave discharges.(ABSTRACT TRUNCATED AT 400 WORDS)


Chemical models of epilepsy with some reference to their applicability in the development of anticonvulsants.

This paper reviews chemical models of epilepsy and their relevance in the identification and characterization of anticonvulsants. For each convulsant we discuss possible modes of administration, clinical type(s) of seizures induced, proposed mechanism(s) of epileptogenesis and, where available, responsiveness of the induced seizures to anticonvulsants. The following compounds are reviewed: pentylenetetrazol, bicuculline, penicillin, picrotoxin, beta-carbolines, 3-mercaptopropionic acid, hydrazides, allylglycine; the glycine antagonist strychnine; gamma-hydroxybutyrate; excitatory amino acids (glutamate, aspartate, N-methyl-D-aspartate, quisqualate, kainate, quinolinic acid); monosubstituted guanidino compounds, metals (alumina, cobalt, zinc, iron); neuropeptides (opioid peptides, corticotropin releasing factor, somatostatin, vasopressin); cholinergic agents (acetylcholine, acetylcholinesterase inhibitors, pilocarpine); tetanus toxin; flurothyl; folates; homocysteine and colchicine. Although there are a multitude of chemical models of epilepsy, only a limited number are applied in the routine screening of potential anticonvulsants. Some chemical models have a predictive value with regard to the clinical profile of efficacy of the tested anticonvulsants. Some chemical models may contribute to a better understanding of possible mechanisms of epileptogenesis.


Valproic acid treatment of experimental status epilepticus.

The efficacy of valproic acid (VPA) in control of generalized convulsive status epilepticus was tested in a rat model. Rats with cortical cobalt lesions were injected with homocysteine thiolactone to induce secondarily generalized tonic-clonic seizures (GTCS). The median effective dose (ED50) for control of GTCS was 211.9 mg/kg (270 micrograms/ml in serum 30 min post dose) when treatment was given intraperitoneally after the second GTCS. VPA entered both serum and brain very rapidly after injection, with little change in concentration from 5 to 30 min post dose. In earlier experiments with phenytoin, phenobarbital, diazepam and lorazepam in this model, we found that the serum concentrations produced by the ED50s versus GTCS were very similar to those which have been reported to be effective in treating human status epilepticus. If this same relationship holds true for VPA, we would predict that a serum concentration of around 270 micrograms/ml VPA would be required for control of generalized convulsive status epilepticus in human patients. The safety of this high a concentration of VPA has not been tested.


Effects of antiepileptic drugs on 4-aminopyridine-induced epileptiform activity in young and adult rat hippocampus.

Extracellular field potential recordings were used to study the effects of the antiepileptic drugs (AEDs) carbamazepine (CBZ), phenytoin (PHT), phenobarbital (PhB) and valproic acid (VPA) on the epileptiform activity evoked by 4-aminopyridine (4-AP, 50 microM) in the CA3 subfield of rat hippocampal slices obtained from young (8-23-day-old) and adult (> 60-day-old) male rats. Ictal (duration: 3-20 s; rate of occurrence: 3-12 x 10(-3) s-1) and interictal (duration: 0.2-0.8 s; rate of occurrence: 0.2-0.8 s-1) discharges were recorded in young slices, while only interictal activity (duration: 70-90 ms; rate of occurrence: 0.5-0.9 s-1) discharges were observed in adult slices. In addition, in both young and adult slices 4-AP disclosed a synchronous long-lasting potential (duration and rate of occurrence: 0.6-3 s, 7-70 x 10(-3) s-1 and 260-660 ms, 8-60 x 10(-3) s-1, respectively) that was caused by the activation of the gamma-aminobutyric acid type A (GABAA) receptor. In young slices, ictal discharges were blocked by CBZ (0.05 mM), PHT (0.1 mM), PhB (0.5 mM) and VPA (0.5 mM). With the exception of PhB, higher concentrations were necessary in these experiments for blocking the interictal activity (i.e., CBZ: 0.1 mM; PHT: > 0.2 mM; VPA: 2 mM). At these concentrations, none of the AEDs blocked the interictal activity in the adult hippocampus, but only reduced the rate of occurrence. PhB enhanced the rate of occurrence of the synchronous GABA-mediated long-lasting potentials both in young (increase: 190%) and in adult (increase: 145%) slices, while VPA increased their occurrence by 54% only in young slices. CBZ decreased the rate of occurrence of this long-lasting potential only in adult hippocampus. Our data indicate that the effects of the AEDs on 4-AP-induced epileptiform discharges are both pattern- and age-dependent. The rank order of potencies of the four AEDs was: (a) in young: CBZ > PHT > PhB > VPA; (b) in adult: CBZ > PhB > PHT > VPA.


Anticonvulsant profile of MDL 27,266: an orally active, broad-spectrum anticonvulsant agent.

The novel anticonvulsant substance MDL 27,266 was tested in a variety of anticonvulsant models to assess its anticonvulsant profile, behavioral toxicity and oral bioavailability. Intraperitoneally (i.p.) administered MDL 27,266 afforded complete protection against sound-induced seizures in DBA/2J and Frings audiogenic-seizure (AGS)-susceptible mice (ED50s: 5.0 and 5.1 mg/kg, respectively). It was also effective following i.p. administration to CF#1 mice against maximal electroshock (MES)-, pentetrazole-, picrotoxin-, quisqualic acid-, and strychnine-induced seizures (ED50s: 24.9, 13.8, 43.3, 8.05, and 60.5 mg/kg, respectively). MDL 27,266, in well tolerated oral doses, prevented the expression of stage 5 behavioral seizures in the corneal-kindled rat and myoclonic seizures in the photosensitive baboon, Papio papio. Chronic administration of MDL 27,266 to AGS-susceptible mice did not markedly affect its anticonvulsant potency or efficacy against sound-induced seizures. These results suggest that MDL 27,266 possesses a broad anticonvulsant profile which most closely approximates that of the broad-spectrum prototype antiepileptic drug valproate.


Cerebrovascular responses to pentylenetetrazol: time and dose dependent effects.

The effects of subconvulsant and convulsant doses of pentylenetetrazol (PTZ) on cerebral blood flow (rCBF), permeability-capillary surface area products (rPS), and brain vascular spaces (BVS) were examined in 15 brain regions at 1 h, 24 h and 1 week after injection in male Sprague-Dawley rats. Brain histology was examined 3 days after injection. A dose of PTZ (50 mg/kg, i.p.), sufficient to trigger a single convulsive seizure, produced small regional changes in rCBF at 1 h, but not at 24 h or 1 week after injection. No significant changes in rPS or BVS were found at any time, and only mild histologic changes were observed. In contrast, a dose of PTZ (25 mg/kg) which failed to cause either convulsions or significant electrocorticographic changes, markedly increased rCBF and rPS. Some of these regional effects were still observed 1 week later. Similarly, more severe and extensive cellular changes followed treatment with the subconvulsive dose. These findings indicate that PTZ treatment can have prolonged effects on cerebrovascular functions and neuronal integrity even in the absence of convulsive activity.


Neuropathological changes during generalized seizures in newborn monkeys.

The brains of four 2-week-old marmoset monkeys were perfusion-fixed immediately after bicuculline-induced seizures lasting 1.5-4.3 h and were later examined by light and electron microscopy. Mean arterial blood pressure and rectal temperature measurements during seizures did not differ significantly from baseline. Plasma glucose concentrations decreased to the 1.5 mM range at the end of seizures, and arterial pH and bicarbonate were lower than in control animals, although arterial pO2 and pCO2 were maintained. Neuropathological changes were minimal. Swollen astrocytic processes surrounded some capillaries and some neurons in cerebral cortex, hippocampus, putamen and thalamus. Almost all the neurons examined looked normal, but mitochondrial swelling was present in a few. All but the most severe mitochondrial swelling, which occurred very rarely in one of four animals, is potentially reversible. The virtual absence of neuronal necrosis in these neonatal monkeys is consistent with the resistance to seizure-induced brain damage found in immature rats, and stands in sharp contrast to the damage seen in older animals. Lack of neuronal damage, however, does not rule out potential adverse effects of prolonged seizure activity on subsequent brain growth and development.


A compound score for estimating the influence of inattention and somnolence during the intracarotid amobarbital test.

Alterations in the level of consciousness may render the interpretation of the memory test results from the intracarotid amobarbital procedure difficult. The present study was designed to investigate the impact of inattention and somnolence on memory performance during the Amytal test. Nineteen consecutive patients undergoing the test were investigated. The memory test was constructed to comprise two consecutive parts with identical design, so as to make possible comparisons over time in the same patients. Reaction level and somnolence were continuously assessed during the procedure and a stimulus-task response test to evaluate the degree of attention was used. On the basis of these parameters a compound 'inattention score' was constructed. The results indicate that inattention and somnolence negatively influence memory performance and should be taken into account when evaluating the Amytal memory test results. In cases with poor memory results high inattention scores may speak in favour of preoperative hemisphere memory testing or a repeat injection with reduced Amytal dosage before deciding upon the extent of a planned resection. On the other hand, low inattention scores together with amnesia for the testing procedure may indicate that the memory test results can be relied on.


Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter.

In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal-binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide-containing phytochelatin-cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1-expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue-specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants.


Primary and secondary structure of the pore-forming peptide of pathogenic Entamoeba histolytica.

A pore-forming peptide is implicated in the potent cytolytic activity of pathogenic Entamoeba histolytica. Using NH2-terminal sequence information of this peptide, the corresponding cDNA was isolated. The cDNA-deduced amino acid sequence revealed a putative signal peptide and a mature peptide of 77 amino acids including six cysteine residues. Computer-aided secondary structure analysis predicted that the peptide would be composed of four adjacent alpha-helices, and CD spectroscopy indicated an all alpha-helical conformation. The tertiary structure appears to be stabilized by three disulfide bonds; the pore-forming activity was not sensitive to heat but was lost in the presence of reducing agents. Sequence homology was found to the saposins and to surfactant-associated protein B, both mammalian polypeptides of similar size and secondary structure but of non-lytic function. In particular, the six cysteine residues were found to be conserved, suggesting a common motif for stabilizing a favourable tertiary structure. Compared with previously characterized toxic peptides also containing three disulfide bonds, the amoeba peptide may represent a distinct class of biologically active peptides.


Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity.

Heat treatment and various other stresses render tumor cells resistant to cytotoxicity mediated by tumor necrosis factors (TNFs). Here, we elucidate the molecular basis of this phenomenon by demonstrating that the major heat shock protein, hsp70, protects tumor cells from TNF cytotoxicity even in the absence of stress. The human hsp70 gene was stably introduced into highly TNF-sensitive WEHI-S tumor cells both in the sense and antisense orientation. All clones constitutively expressing the exogenous human hsp70 gene were protected from TNF-mediated killing approximately 1000-fold. Remarkably, the growth of one clone was actually stimulated by low concentrations of TNF. Moreover, a clone expressing antisense hsp70 RNA was rendered extremely sensitive to TNFs. Hsp70-mediated protection from TNF cytotoxicity was confirmed in transient expression experiments employing retroviral vectors. Changes in cellular sensitivity to TNF were not associated with alterations in the binding of TNF to its receptors. Neither the transfection procedure itself nor overexpression of the low molecular weight heat shock protein, hsp27, had any effect on cellular susceptibility to TNFs. Our data suggest that hsp70 may increase the oncogenic potential of some tumor cells by providing them with an escape mechanism from immunological defense.


Interaction of heat-shock protein 70 with p53 translated in vitro: evidence for interaction with dimeric p53 and for a role in the regulation of p53 conformation.

In intact cells, hsp70 proteins selectively complex with mutant p53. We report here that rabbit reticulocyte lysate contains hsp70 which selectively complexes with the mutant p53 translated in vitro. Hsp70 complexes with dimers and possibly monomers of p53 in a manner that requires the terminal 28 amino acids of p53. Using murine p53Val135, which is temperature-sensitive for phenotype, we demonstrate that p53-hsp70 complexes can occur after post-translational switching from wild-type to mutant p53 phenotype. Moreover, the temperature-induced switch of full-length p53Val135 from wild-type to mutant phenotype is ATP-independent, whereas the switch from mutant to wild-type form requires ATP hydrolysis and involves hsp70. These results imply that hsp70 is involved in the regulation of p53 conformation.


Critical cytoplasmic domains of the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth signal transduction and tyrosine phosphorylation.

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 5 (IL-5) are composed of two distinct subunits, alpha and beta c. The alpha subunits are specific for each cytokine, whereas the beta subunit (beta c) is shared by the three receptors and is an essential component of signal transduction. We have made a series of mutant beta c cDNAs that delete various regions of the cytoplasmic domain and examined the function of these mutants by coexpressing them with the alpha subunit of the human GM-CSF receptor (hGMR) in an IL-3-dependent mouse pro-B cell line BaF3. Two domains in the membrane-proximal portion of beta c were found to be important for transducing the hGM-CSF-mediated growth signals: one domain between Arg456 and Phe487 appears to be essential for proliferation, and the second domain between Val518 and Asp544 enhances the response to GM-CSF, but is not absolutely required for proliferation. The region between Val518 and Leu626 was responsible for major tyrosine phosphorylation of 95 and 60 kDa proteins. Thus, beta c-mediated major tyrosine phosphorylation of these proteins was apparently separated from proliferation. However, the beta 517 mutant lacking residues downstream of Val518 transmitted a herbimycin-sensitive proliferation signal, suggesting that beta 517 still activates a tyrosine kinase(s). We also evaluated the role of the cytoplasmic domain of the GMR alpha subunit and the results suggest that it is involved in the hGM-CSF-mediated signal transduction, but is not essential.(ABSTRACT TRUNCATED AT 250 WORDS)


Signal transduction between enteropathogenic Escherichia coli (EPEC) and epithelial cells: EPEC induces tyrosine phosphorylation of host cell proteins to initiate cytoskeletal rearrangement and bacterial uptake.

Upon attachment to cultured HeLa cells, enteropathogenic Escherichia coli (EPEC) induces assembly of a complex cytoskeletal structure within the eucaryotic cell, localized beneath the adherent bacterium. In addition, EPEC induces its own internalization by non-phagocytic epithelial cells. We found that after binding to the epithelial cell surface, EPEC induces tyrosine phosphorylation of three eucaryotic proteins. The major phosphorylation substrate is a 90 kDa protein (Hp90). In correlation with Hp90 tyrosine phosphorylation, the EPEC-induced cytoskeletal structure also contained tyrosine phosphorylated proteins. Using tyrosine protein kinase inhibitors and EPEC mutants (cfm) that fail to induce Hp90 phosphorylation, we demonstrate that induction of Hp90 phosphorylation is involved in initiation of the cytoskeletal structure assembly and in bacterial uptake. Other non-invasive EPEC mutants (eae) are still able to induce Hp90 tyrosine phosphorylation and to initiate aggregation of the tyrosine phosphorylated proteins and some cytoskeleton components. However, eae mutants are deficient in nucleating the aggregates into an organized structure.


Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center.

Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.


Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.

Tetanus and botulinum neurotoxins are the most potent toxins known. They bind to nerve cells, penetrate the cytosol and block neurotransmitter release. Comparison of their predicted amino acid sequences reveals a highly conserved segment that contains the HexxH zinc binding motif of metalloendopeptidases. The metal content of tetanus toxin was then measured and it was found that one atom of zinc is bound to the light chain of tetanus toxin. Zinc could be reversibly removed by incubation with heavy metal chelators. Zn2+ is coordinated by two histidines with no involvement in cysteines, suggesting that it plays a catalytic rather than a structural role. Bound Zn2+ was found to be essential for the tetanus toxin inhibition of neurotransmitter release in Aplysia neurons injected with the light chain. The intracellular activity of the toxin was blocked by phosphoramidon, a very specific inhibitor of zinc endopeptidases. Purified preparations of light chain showed a highly specific proteolytic activity against synaptobrevin, an integral membrane protein of small synaptic vesicles. The present findings indicate that tetanus toxin, and possibly also the botulinum neurotoxins, are metalloproteases and that they block neurotransmitter release via this protease activity.


Developmental modification of lipophosphoglycan during the differentiation of Leishmania major promastigotes to an infectious stage.

Protozoan parasites of the genus Leishmania produce the novel surface glycoconjugate, lipophosphoglycan (LPG), which is required for parasite infectivity. In this study we show that LPG structure is modified during the differentiation of L. major promastigotes from a less infectious form in logarithmic growth phase to a highly infectious 'metacyclic' form during stationary growth phase. In both stages, the LPGs comprise linear chains of phosphorylated oligosaccharide repeat units which are anchored to the membrane via a glycosyl-phosphatidylinositol glycolipid anchor. During metacyclogenesis there is (i) an approximate doubling in the average number of repeat units per molecule from 14 to 30, (ii) a pronounced decrease in the relative abundance of repeat units with side chains of beta Gal or Gal beta 1-3Gal beta 1-, and a corresponding increase in repeat units with either no side chains or with side chains of Arap alpha 1-2 Gal beta 1- and (iii) a decrease in the frequency with which the glycolipid anchor is substituted with a single glucose alpha 1-phosphate residue. While the majority of the LPG phosphoglycan chains are capped with the neutral disaccharide, Man alpha 1-2Man, a significant minority of the chains appeared to terminate in non-phosphorylated repeat units and may represent incompletely capped species. We suggest that the developmental modification of LPG may be important in modulating the binding of promastigotes to receptors in the sandfly midgut and on human macrophages and in increasing the resistance of metacyclic promastigotes to complement-mediated lysis.


A novel antifolate resistance gene on the amplified H circle of Leishmania.

In several Leishmania spp., resistance to methotrexate and other drugs is often associated with amplification of the chromosomal H region in the form of extrachromosomal H circles. We report here that the H circle of Leishmania tarentolae contains an 867 bp open reading frame, ltdh, which mediates high levels of resistance to methotrexate and other antifolates, after transfection. The predicted amino acid sequence of the ltdh gene product has significant similarities to a family of short-chain dehydrogenases, enzymes that are involved in several oxido-reduction reactions in a wide range of organisms. To resist antifolates, Leishmania amplifies the ltdh gene as part of the H circle. We propose that LTDH might be involved in an alternative pathway for the synthesis of reduced folates and that ltdh overproduction represents a novel mechanism for resistance to antifolates. Our results support the hypothesis that the H region of the Leishmania genome contains several drug resistance genes and that preferential amplification of this region has evolved as a defense mechanism against cytotoxic drugs.


Bos1p, a membrane protein required for ER to Golgi transport in yeast, co-purifies with the carrier vesicles and with Bet1p and the ER membrane.

BOS1 and BET1 are required for transport from the ER to the Golgi complex in yeast and genetically interact with each other and a subset of the other genes, whose products function at this stage of the secretory pathway. In a previous study, we reported that BOS1 encodes a putative 27 kDa membrane protein. Here we show that BET1 is structurally similar to the synaptobrevins and identical to the SLY12 gene product. Overexpression of SLY12 compensates for the loss of function of the ras-like GTP-binding protein Ypt1. Both Bos1p and Bet1p are cytoplasmically oriented membrane proteins. Bos1p co-purifies with the ER to Golgi transport vesicles and co-fractionates with Bet1p and the ER membrane.


MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria.

To identify components of the mitochondrial protein import pathway in yeast, we have adopted a positive selection procedure for isolating mutants disturbed in protein import. We have cloned and sequenced a gene, termed MPI1, that can rescue the genetic defect of one group of these mutants. MPI1 encodes a hydrophilic 48.8 kDa protein that is essential for cell viability. Mpi1p is a low abundance and constitutively expressed mitochondrial protein. Mpi1p is synthesized with a characteristic mitochondrial targeting sequence at its amino-terminus, which is most probably proteolytically removed during import. It is a membrane protein, oriented with its carboxy-terminus facing the intermembrane space. In cells depleted of Mpi1p activity, import of the precursor proteins that we tested thus far, is arrested. We speculate that the Mpi1 protein is a component of a proteinaceous import channel for translocation of precursor proteins across the mitochondrial inner membrane.


DNA transcription and repressor binding affect deletion formation in Escherichia coli plasmids.

Chimeric plasmids containing phage M13 and plasmid pBR322 sequences undergo deletions in Escherichia coli with a high frequency. In all plasmids one deletion endpoint is the M13 replication origin nick site. We examined the effects of transcription on the position of the other deletion end-point, by inserting in the plasmids an inducible promoter followed by a transcription terminator. Transcription dramatically affected deletions in an orientation-dependent way, such that greater than 95% of end-points were localized downstream from the inserted promoter when it faced the major plasmid transcripts. The end-points were not constrained to the transcribed region and were not affected by the orientation of pBR322 DNA replication. We propose that deletion events occur preferentially in a plasmid domain which is rendered positively supercoiled by convergent transcription. We also show that interaction of LacI repressor with the cognate operator generates a localized deletion hot spot. This hot spot is dependent on pBR322 replication, and therefore probably acts by arresting progression of DNA replication.


Trans regulation in the Ultrabithorax gene of Drosophila: alterations in the promoter enhance transvection.

We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.


Mouse rRNA gene transcription factor mUBF requires both HMG-box1 and an acidic tail for nucleolar accumulation: molecular analysis of the nucleolar targeting mechanism.

RNA polymerase I requires at least two nucleolar transcription factors, UBF and SL-1, for ribosomal RNA gene (rDNA) transcription. UBF requires SL-1 for the formation of a stable initiation complex on the rDNA promoter region. We have determined the region of mouse UBF (mUBF) required for nucleolar targeting. Although mUBF has a nuclear localization sequence, this sequence alone is not sufficient for mUBF to accumulate in the nucleolus. Deletion analyses show that mUBF requires a wide region except for the N-terminal 101 amino acids for nucleolar targeting. Deletion of either the HMG-box1, a region crucial for rDNA binding, or the acidic tail, a region that may interact with SL-1, results in the loss of nucleolar targeting. We show by DNA affinity analysis that the HMG-box1 is absolutely necessary for mUBF to bind to the upstream control element of the rDNA. We also show that mUBFs with various internal deletions retain both nucleolar targeting and DNA binding ability. A clear correlation was demonstrated between the DNA binding and nucleolar targeting ability. These results suggest that UBF is transferred to the nucleus by its NLS and is sequestered in the nucleolus by its specific and stable binding to the rDNA promoter via HMG-boxes and the acidic tail.


An SRY-related gene expressed during spermatogenesis in the mouse encodes a sequence-specific DNA-binding protein.

SRY, the testis determining gene, encodes a member of a family of DNA binding proteins characterized by an amino acid sequence motif known as the HMG box. Using degenerate primers and the polymerase chain reaction, we have isolated SRY-related cDNAs from adult murine testis RNA. One of these, Sox-5, encodes a 43 kDa HMG-box protein with similarities to transcription activating proteins. Anti-Sox-5 antibody was used to analyse expression of Sox-5 in pre-pubertal testis and in fractionated spermatogenic cells. Sox-5 is restricted to post-meiotic germ cells, being found at highest levels in round spermatids. Sox-5 is a DNA binding protein and binding site selection assays suggest that it can bind specifically to oligonucleotides containing the consensus motif AACAAT. Sry can also bind to this motif, indicating that the Sry family may have overlapping sequence specificities.


Roles of PRP8 protein in the assembly of splicing complexes.

Three different approaches have been used to investigate the roles of the yeast U5 snRNP protein PRP8 in spliceosome assembly: genetic depletion of PRP8 protein in vivo, heat inactivation of temperature-sensitive prp8 protein in protoplasts and inhibition of PRP8 function with antibodies in vitro. In each case, U5 and U4/U6 snRNPs failed to assemble into the forming spliceosomes. In addition, extract prepared from PRP8-depleted cells and extract containing inactivated PRP8 protein had substantially reduced amounts of U4/U6.U5 triple snRNP complexes. Thus, functional PRP8 protein is required for the stable formation of U4/U6.U5 complexes without which spliceosomes fail to form. As spliceosome formation was also blocked by anti-PRP8 antibodies that apparently do not disrupt triple snRNPs, PRP8 protein may play a separate role in the assembly of triple snRNPs into spliceosomes. As a consequence of PRP8 depletion the levels of the U4, U5 and U6 snRNAs declined dramatically. We discuss this in the context of the known genetic interactions between PRP8 and putative RNA helicase (DEAD box protein) genes and propose that PRP8 protein plays a role in regulating dynamic RNA-RNA interactions in spliceosome assembly, possibly ensuring the correct directionality of these events.


A yeast splicing factor is localized in discrete subnuclear domains.

Digital imaging microscopy has been used to visualize the splicing protein PRP6p and three other yeast nuclear proteins. The results show that PRP6p is uniquely localized to discrete subnuclear regions. A combination of cytological and biochemical assays suggests that these sites can be saturated when the protein is overexpressed and likely correspond to the location of U4/U6 snRNPs. The observations indicate that some splicing components are located in discrete subregions of the yeast nucleus, similar to the situation described for the mammalian nucleus.


Coding from a distance: dissection of the mRNA determinants required for the incorporation of selenocysteine into protein.

Incorporation of selenocysteine into proteins is directed by specifically 'programmed' UGA codons. The determinants for recognition of the selenocysteine codon have been investigated by analysing the effect of mutations in fdhF, the gene for formate dehydrogenase H of Escherichia coli, on selenocysteine incorporation. It was found that selenocysteine was also encoded when the UGA codon was replaced by UAA and UAG, provided a proper codon-anticodon interaction was possible with tRNA(Sec). This indicates that none of the three termination codons can function as efficient translational stop signals in that particular mRNA position. The discrimination of the selenocysteine 'sense' codon from a regular stop codon has previously been shown to be dependent on an RNA secondary structure immediately 3' of the UGA codon in the fdhF mRNA. It is demonstrated here that the correct folding of this structure as well as the existence of primary sequence elements located within the loop portion at an appropriate distance to the UGA codon are absolutely required. A recognition sequence can be defined which mediates specific translation of a particular codon inside an mRNA with selenocysteine and a model is proposed in which translation factor SELB interacts with this recognition sequence, thus forming a quaternary complex at the mRNA together with GTP and selenocysteyl-tRNA(Sec).


Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure.

In order to test the proposal that most nucleotide polymerases share a common active site structure and folding topology, we have generated 22 mutations of residues within motifs A, B and C of T7 RNA polymerase (RNAP). Characterization of these T7 RNAP mutants showed the following: (i) most of the mutations resulted in moderate to drastic reductions in T7 RNAP transcriptional activity supporting the idea that motifs A, B and C identify part of the polymerase active site; (ii) the degree of conservation of an amino acid within these motifs correlated with the degree to which mutation of that amino acid reduced transcriptional activity, supporting the predictive ability of this alignment in identifying the most functionally critical residues; (iii) a comparison of DNAP I and T7 RNAP mutants revealed similarities (as well as differences) between corresponding mutant phenotypes; (iv) the Klenow fragment structure is shown to provide a reasonable basis for interpretation of the differential effects of mutating different amino acids within motifs A, B and C in T7 RNAP. These observations support the proposal that these polymerase active sites have similar three-dimensional structures.


Alternating d(G-A) sequences form a parallel-stranded DNA homoduplex.

The oligonucleotides d[(G-A)7G] and d[(G-A)12G] self-associate under physiological conditions (10 mM MgCl2, neutral pH) into a stable double-helical structure (psRR-DNA) in which the two polypurine strands are in a parallel orientation in contrast to the antiparallel disposition of conventional B-DNA. We have characterized psRR-DNA by gel electrophoresis, UV absorption, vacuum UV circular dichroism, monomer-excimer fluorescence of oligonucleotides end-labelled with pyrene, and chemical probing with diethyl pyrocarbonate and dimethyl sulfate. The duplex is stable at pH 4-9, suggesting that the structure is compatible with, but does not require, protonation of the A residues. The data support a model derived from force-field analysis in which the parallel-stranded d(G-A)n helix is right-handed and constituted of alternating, symmetrical Gsyn.Gsyn and Aanti.Aanti base pairs with N1H...O6 and N6H...N7 hydrogen bonds, respectively. This dinucleotide structure may be the source of a negative peak observed at 190 nm in the vacuum UV CD spectrum, a feature previously reported only for left-handed Z-DNA. The related sequence d[(GAAGGA)4G] also forms a parallel-stranded duplex but one that is less stable and probably involves a slightly different secondary structure. We discuss the potential intervention of psRR-DNA in recombination, gene expression and the stabilization of genomic structure.