Image analysis of comet assay measurements

In the last decade the 'comet assay' or 'single cell gel electrophoresis assay' has been established as a sensitive method for the detection of DNA damage and the measurement of its recovery. The results published in the literature have often been obtained with different methods for comet structure measurement. In most cases these data are not comparable with each other. Even when using similar systems for the analysis, it is difficult to obtain matching data. This presentation will describe some technical aspects of our measurement equipment and evaluation software. It focuses on necessary experimental conditions to minimize errors in obtaining such data. The software developed here allows the rapid analysis of the microscopic samples (< 2 s per image). The image analysis was designed with respect to the morphological shapes of comet cells, which were investigated with a confocal laser microscope. The system is built with standard components which are commercially available. As a measure of the amount of DNA damage the ratio of fluorescence intensity was used inside the comet tail and the fluorescence intensity of the comet head. Other parameters such as DNA content, comet area, head radius, tail length and tail moment are also determined. The reproducibility of the system has been evaluated in several experiments over a period of 5 years.     

Co-localization of serotonin and FMRFamide-like immunoreactivities in the central nervous system of the earthworm, Eisenia fetida

In addition to receptor-type pinealocytes, the mammalian pineal organ contains small and large neurons and ependymal/glial cells as well. Axons of pinealocytes form synaptic ribbon-containing axo-dendritic synapses on large secondary pineal neurons and/or terminate as neurohormonal endings on the basal lamina of the vascular surface of the organ. The small pineal neurons were found to be gamma-aminobutyric acid (GABA)-immunoreactive, while large secondary neurons and pinealocytes contained immunoreactive amino acids (glutamate and aspartate). Glutamate accumulated presynaptically in pinealocytic axon terminals on large secondary neurons and in the axons of these neurons. Glutamate immunoreactive axons of pineal neurons were traced via the pineal tract to the habenular nucleus. Axons containing granular vesicles and coming from extrapineal perikarya are glutamate immunoreactive as well. Aspartate and GABA are also present in some of the myelinated axons, supposedly pinealopetal in the pineal tract.     

Mediastinal space-occupying lesion with reduced perfusion of the left lung. Thoracic aortic aneurysm with compression of the left pulmonary artery

CONTEXT: An increase in the recreational use of personal watercraft (PWC) raises concern about an increase in associated injuries on a national level. OBJECTIVE: To estimate the relative frequency, types of injury, and demographic features of persons injured while using PWC in the United States. DESIGN: Case series. SETTING: Emergency department (ED) visits to hospitals participating a national probability sample. PARTICIPANTS: All persons treated for PWC-related injury from January 1,1990, through December 31, 1995. RESULTS: An estimated 32954 persons (95% confidence interval [CI], 22919-42989) with PWC-related injuries were treated in US hospital EDs, of which 3.5% were hospitalized. Personal watercraft-related injuries have increased significantly from an estimated 2860 in 1990 to more than 12000 in 1995. During this period, the number of PWC in operation increased 3-fold from approximately 241500 in 1990 to an estimated 760000 in 1995. The most prevalent diagnoses were lacerations, contusions, and fractures. MAIN OUTCOME MEASURES: The estimated number and percentage of patients treated in EDs for PWC-related injuries, by year, age, sex, and the number and rate per 1000 of PWC in operation by year. CONCLUSIONS: Since 1990, there has been at least a 4-fold increase in injuries associated with an increase in the recreational use of PWC. The rate of ED-treated injuries related to PWC was about 8.5 times higher (95% CI, 8.2-8.8; 1992 data) than the rate of those from motorboats. Specific training and adult supervision is recommended for minors using PWC. Furthermore, medical practitioners should encourage personal flotation device use and other protection for their patients who are known water enthusiasts.     

Fibrinolysis, antithrombin III, and protein C in neonates during cardiac operations

Fibrinolysis and coagulation were studied in 10 neonates undergoing cardiac operations for congenital heart defects. Coagulation was activated during cardiopulmonary bypass as evidenced by highly increased prothrombin fragment 1 + 2 levels compared with preoperative values. Prothrombin fragment 1 + 2 levels remained elevated until postoperative day 3. Unlike coagulation, fibrinolysis was not activated during cardiopulmonary bypass but did show late activation on postoperative day 3, as evidenced by elevated levels of the fibrin degradation product D-dimer. Lack of fibrinolytic activation during bypass and its appearance on postoperative day 3 were partly explained by changes observed in tissue plasminogen activator and its inhibitor. During bypass, levels of tissue plasminogen activator and its inhibitor increased by 3.4-fold and 3.2-fold, respectively. In the postoperative period, levels of plasminogen activator inhibitor normalized rapidly whereas tissue plasminogen activator remained elevated, resulting in late fibrinolytic activation on postoperative day 3. In accordance with elevated prothrombin fragment 1 + 2, platelet count, antithrombin III, protein C, prothrombin, and factor VII were decreased on postoperative day 2, indicating ongoing consumptive coagulopathy. Nine patients had antithrombin III and six had protein C levels below age-specific normal ranges, consistent with an acquired deficiency state. Three had central venous thrombosis by postoperative day 4 or 5. In all three, thrombosis was preceded by antithrombin III deficiency, protein C deficiency, and highly elevated plasminogen activator inhibitor (3.7 to 37 times the mean of the other patients) on postoperative days 1 to 3. In conclusion, cardiopulmonary bypass in neonates caused rapid and profound alterations in the coagulation and fibrinolytic systems and initiated consumptive coagulopathy lasting until at least postoperative day 3. Thrombophilic abnormalities in antithrombin III, protein C, and fibrinolysis were frequently found and were associated with serious thrombotic complications.     

Pathogenetic aspects of the L-arginine-NO metabolic pathway in arteriosclerosis and possible therapeutic aspects

A yearly pattern in the occurrence of uterine cervical cancer (UCC), obtained from cytological examinations reported as type V (cases concluding a malignant alteration), has been previously shown for data obtained in the Monterrey Metropolitan Area (state of Nuevo Leon, Mexico) for a span of 10 years (1978-1987), with a peak of relative incidence in the month of February being high stable for consecutive years. With the aim of extending and validating those results, we analyzed the monthly totals of positive detected cases of UCC in the states of Nuevo Leon, Chihuahua, Coahuila, and Tamaulipas (covering most of Northern Mexico) during the same period. To eliminate bias due to the seasonal variation in the number of screening smears, data were first expressed in relation to the number of cytological examinations done the same month. The least-squares fit of a 1-year cosine curve to the data of relative incidence in the four states reveals a statistically significant yearly pattern (p = .008), with a maximum of relative incidence in February almost double that during the rest of the year. Results indicate that the relative incidence of UCC is higher than the yearly average during the winter, with secondary peaks in May and October. In view of the nonsinusoidal waveform in the incidence of UCC, we undertook a multiple-component analysis, allowing several cosine functions to be simultaneously fitted to the data. Results indicate that the yearly pattern in the relative incidence of UCC can be represented by a model that includes two components with periods of 12 and 4 months (p = .004). The same model can be documented as statistically significant independently for each of the four states. These results, summarizing over 2200 positive cases of UCC detected in more than 1,100,000 screening smears, are in full agreement with those found previously for part of the state of Nuevo Leon and reveal a highly stable and predictable yearly pattern of variation in the relative incidence of UCC in Northern Mexico.     

Interaction of Yttria with Uranium Fluoride Gases (UFn, n= 4, 6) at High Temperatures

The corrosion mechanism and microstructural characteristics of yttria (Y₂O₃) exposed to uranium hexafluoride (UF₄) and uranium tetrafluoride gases at temperatures above 900°C were investigated. Processed yttria samples were exposed for times ranging from 5 to 40 min. A weight increase was observed after each test exposure. An outer corrosion scale composed of UO₂ and YF₃, and an inner corrosion layer of YF₃, were observed following the UF₆ reaction at nearly 1200 K. During the UF₄ exposure, an outer layer containing a liquid mixture of fluoride, oxygen, yttrium, uranium compounds, a secondary reaction layer of UO₂ followed by an inner layer composed of a solution of Y₂O₃ and YF₃, possibly in the form of YOF inner layer, were observed. It was found that the corrosion process is controlled by the diffusion which is evidenced by the parabolic growth rate of the secondary layer. After completion of the solidification process, the formation of a eutectic region and dendrites were observed throughout the outer scale. In general, at temperatures above 1173 K, high corrosion rates were observed, and yttria did not appear to be a viable containment material for UF₄ and UF₆.     

Mechanical Properties of Large Arteries in Mother and Fetus during Normal and Diabetic Pregnancy

Cardiodilatin (CDD)/atrial natriuretic peptide (ANP) is a 28-amino acid peptide hormone known to be synthesized in the heart of a large number of different vertebrates. It plays an important role in the regulation of blood pressure and natriuresis/diuresis. Since the cardiovascular system of the horse has to meet the highest requirements concerning its physiological performance, we intended to characterize the cardiodilatin/atrial natriuretic peptide system of this species. By means of immunohistochemistry and immunoelectron microscopy, we precisely identified auricular cardiocytes as the loci of CDD/ANP synthesis. Using aortic smooth muscle relaxation assay and CDD/ANP-ELISA, we succeeded in isolating the biologically active prohormone. We subsequently cloned the equine cDNA of the CDD/ANP precursor protein and deduced its primary sequence. The entire precursor protein is in good agreement with the CDD/ANP prohormones of other mammals. The deduced theoretical average Mr of equine CDD/ANP-1-126 is 13,764, corresponding to the molecular weight of purified peptide determined by ESI-MS. Our findings suggest that equine CDD/ANP is produced in auricular cardiocytes and the predominant storage form of CDD/ANP in the auricle is the prohormone CDD/ANP-1-126.     

Detection and identification of mycobacteria in formalin-fixed, paraffin-embedded tissues by nested PCR and restriction enzyme analysis

A novel assay based on a nested PCR and restriction enzyme analysis of the PCR products was developed for the rapid detection and identification of Mycobacterium bovis and M. avium-M. intracellulare species in formalin-fixed, paraffin-embedded tissue (PET) specimens. On the basis of the nucleotide sequence data obtained in the present study, general nested primers were constructed to amplify a 424-bp segment of the gene encoding the 65-kDa surface antigen of mycobacteria. The nested PCR assay proved to be highly sensitive, since as little as 5 to 10 fg of extracted mycobacterial DNA was detected. The safety of the assay as a routine method for the diagnosis of M. bovis and M. avium-M. intracellulare in PET specimens was provided by taking various precautions. In order to prevent false positivity, specific tools and procedures were applied. To detect false-negative results and assess the efficiency of the PCR, an internal standard molecule of amplification was constructed. The digestion of the amplicons with the restriction endonuclease Sau96-I allowed the identification of M. bovis and M. avium-M. intracellulare in a large number of clinical specimens. The present results indicate that PCR combined with an internal control of amplification and restriction enzyme analysis of the amplicons provides a rapid, sensitive, and reliable method for routine diagnostic laboratories to detect and identify M. bovis and M. avium-M. intracellulare in PET specimens.