Optimization and Standardization of the “Comet Assay” for Analyzing the Repair of DNA Damage in Cells

BACKGROUND: The "comet assay" has become an interesting and a very useful tool for the analysis of the induction and amount of DNA damage in single cells thus offering the opportunity to measure the effectiveness of DNA repair. On the basis of the Ostling and Johanson protocol we have developed a modified method with increased sensitivity and high reproducibility. MATERIAL AND METHODS: Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the "comet assay" technique. RESULTS: In this presentation the influences of different methodological factors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the "comet assay" are described in detail and optimum conditions for "comet assay" experiments have been evaluated. Additionally the authors will show a comparison of different fluorescent DNA dyes pointing out their advantages or disadvantages for "comet" analysis. The usefulness of this technique and its capabilities are exemplified by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays. CONCLUSIONS: This paper presents data on the optimization and standardization of the original "comet assay" leading to an extremely fast and practicable protocol in the field of single cell gel electrophoresis. After irradiation with 0.1 Gy an increase in the amount of DNA damage can be measured with high statistical significance and the DNA repair capacity of individual cells after X-ray doses of 2 Gy can be analyzed with high reproducibility. The results comparing DNA repair capacities of different donors point out that the "comet assay" may have the potential for the estimation of individual radiosensitivity.     

Localization of the nonpalpable colonic lesion with intraoperative ultrasound

Localization of an nonpalpable colonic lesion at the time of colectomy usually requires intraoperative colonoscopy. The use of ultrasound to locate the lesion has not been described. A soft bowel clamp is placed above the expected location of the lesion and a catheter placed in the anus. Saline is then instilled into the colon and rectum. The lesion is located by ultrasound scan of the fluid filled colon with the probe placed on the serosal surface. Refinement of the technique was performed on resected colonic specimens. An in vivo trial was then performed with rapid and accurate localization of the lesion for resection. Intraoperative ultrasound of the colon can accurately localize nonpalpable colonic lesions and is an alternative to currently available techniques of localization. Received: 10 December 1997/Accepted: 11 March 1998     

Core beliefs in anorexic and bulimic women

There is research evidence to suggest the presence of dysfunctional cognitions in anorexia nervosa and bulimia nervosa that are not related to food, weight, or shape. These maladaptive cognitions have not been addressed by the conventional cognitive behavioral models of etiology or therapy. This study aimed to assess the impact of unhealthy core beliefs on eating disorders and their symptoms. Twenty restricting anorexics, 10 bulimic anorexics, 27 bulimics, and 23 normal controls completed Young's Schema Questionnaire. Eating behaviors and attitudes were also measured. The results indicate that both anorexic and bulimic women had significantly higher levels of unhealthy core beliefs than comparison women, but the clinical groups only differed on one individual core belief (entitlement). However, there were different patterns of association between core beliefs and eating psychopathology in anorexic and bulimic women. It is suggested that future clinical practice should incorporate core beliefs as a potential element in the assessment and treatment of eating disorders.     

MRI findings in Hirayama’s disease: flexion-induced cervical myelopathy or intrinsic motor neuron disease?

Hirayama’s disease is a benign juvenile form of focal amyotrophy affecting the upper limbs. Previous studies have suggested that the disorder is a neck flexion induced cervical myelopathy. We report clinical and magnetic resonance imaging findings in nine patients with Hirayama’s disease. Cervical imaging of seven patients revealed spinal cord changes consisting of focal atrophy and foci of signal alterations. On neck flexion a forward movement and mild reduction in the anteroposterior diameter of the lower cervical cord against the vertebral bodies was noted in affected individuals as well as in five normal controls. In contrast to earlier reports, none of our patients showed complete obliteration of the posterior subarachnoid space. Measurement of the anteroposterior spinal cord diameter in each vertebral segment (C4–C7) revealed no significant differences in the degree of spinal cord flattening between the two groups. Furthermore, two of our patients had significant degenerative changes in the cervical spine (disc herniation, retrospondylosis) contralateral to the clinically affected side. These degenerative changes resulted in a marked cord compression on neck flexion but were not associated with ipsilateral clinical abnormalities or spinal cord alterations. Our results argue against a flexion-induced cervical myelopathy and support the view that Hirayama’s disease is an intrinsic motor neuron disease. Received: 15 March 1999 Received in revised form: 25 May 1999 Accepted: 1 June 1999     

Motor rehabilitation after traumatic brain injury and stroke - Advances in assessment and therapy

A long-term goal in motor rehabilitation is that treatment is not selected on the basis of 'schools of thought', but rather, based on knowledge about efficacy and effectiveness of specific interventions for specific situations (e.g. functional syndromes). Motor dysfunction after stroke or TBI can be caused by many different functional syndromes such as paresis, ataxia, deafferentaion, visuo-perceptual deficits, or apraxia. Examples are provided showing that theory-based analysis of motor behavior makes it possible to describe 'syndrome-specific motor deficits'. Its potential implications for motor rehabilitation are that our understanding of altered motor behavior as well as specific therapeutic approaches might be promoted. A methodological prerequisite for clinical trials in rehabilitation is knowledge about test properties of assessment tools in follow-up situations such as test-retest reliability and responsiveness to change. Test-retest reliability assesses whether a test can produce stable measures with test repetition, while sensitivity to change reflects whether a test detects changes that occur over time. Exemplifying these considerations, a reliability and validity study of a kinematic arm movement analysis is summarized. In terms of new therapeutic developments, two examples of clinical therapeutic studies are provided assessing the efficacy of specific inter-ventions for specific situations in arm and gait rehabilitation: the Arm Ability Training for high functioning hemiparetic stroke and TBI patients, and the treadmill training for non-ambulatory hemiparetic patients. In addition, a new technical development, a machine-controlled gait trainer ist introduced.     

Cognitive Behavioral Therapy of Minor Depressive Symptoms in Elderly Chinese Americans: A Pilot Study

There is a high prevalence of suicide amongelderly Chinese, and particularly among elderly Chinesewomen in Mainland China with a prevalence of 19.6 perhundred thousand. Since Chinese individuals may much more highly value education, acognitive-behavioral package originated by RicardoMunoz, Ph.D. was adapted for Chinese American subjects.The material was videotaped in eight sessions,approximately 25 minutes in length, to be shown to communitysubjects who were at least 40 years and over. Inaddition, a videotape of muscular relaxation techniqueswas made. A manual written in Chinese about the content of each class, was given to each subject whenhe/she attended. The experimental group showedsignificant improvement in the scores in the HamiltonDepression Scale, including the Somatic Subscale in the Hamilton Anxiety Scale. There was nosignificant improvement in the control group on any ofthe measures. Thus the study suggests the efficacy ofpsychoeducational classes in reducing symptoms ofdepression in non-patient community elderly. Other studiesare being conducted among Korean Americans and JapaneseAmericans in the United States, and also in the Orientamong Japanese elderly.     

A case of agitated catatonia

Agitation is one of the diagnostic features of catatonia in the DSM IV classification, but permanent forms of agitated catatonia have occasionally been described. We report the case of a 43-year-old man who had already suffered from undifferentiated schizophrenia for 7 years, and in whom we diagnosed agitated catatonia. While our patient was being treated with a neuroleptic during a second episode of paranoia, a state of agitation was observed which persisted for a further 8 months. During this period, he was treated with several different neuroleptics and benzodiazepines, either alone or in association, without any improvement. No organic cause was found. He was then transferred to our electroconvulsive therapy (ECT) unit, with a diagnosis of schizophrenic agitation resistant to drug therapy. ECT was begun, and he was only given droperidol in case of agitation and alimemazine for insomnia, neither of which had any effect. In view of his persistent agitation without any purpose, echolalia and echopraxia, stereotyped movements with mannerisms and marked mimicking and grimacing, we diagnosed him as having agitated catatonia. After the fourth session of ECT, we decided to stop all treatment and gave him lorazepam at a dose of 12.5 mg daily. Twenty-four hours later, all symptoms of agitation had disappeared. In our opinion, permanent catatonic agitation is not rare. In our case, the neuroleptic treatment maintained and may even have worsened the symptomatology. Lorazepam can be used as a therapeutic test for this type of agitation, especially if it does not respond to neuroleptics. This also allows the patient to be sedated rapidly and effectively, thus preventing him from injuring himself further.