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.     

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.     

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.     

The anabolic effects of parathyroid hormone on cortical bone mass, dimensions and strength--assessed in a sexually mature, ovariectomized rat model

We examined the effect of the pineal neurohormone melatonin (MLT) on protection from viral encephalitis. The antiviral activity of MLT was evaluated in normal mice inoculated with Semliki Forest virus (SFV) and in stressed mice injected with the attenuated non-invasive West Nile virus (WN-25). Administration of MLT (s.c.) daily from 3 days before through 10 days after virus inoculation reduced viremia and significantly postponed the onset of disease and death by 7 to 10 days. Moreover, MLT injection reduced mortality of SFV (10 PFU) inoculated mice from 100% to 44%. In mice inoculated with high dose of SFV (100 PFU), MLT postponed death and reduced mortality by 20%. In all of the surviving mice anti-SFV antibodies were detected 22 days after virus inoculation. Infection of mice stressed by either isolation or dexamethasone injection with WN-25 induced mortality of 75% and 50% respectively, which was reduced by MLT administration to 31% and 25%, respectively. The efficiency of MLT in protecting from lethal viral infections warrants further investigations on its mechanisms of action.