Biomedical subjects
Publications and source records attributed to C Bock.
Hexamethyldisilazane treatment and subsequent air drying of spread plant chromosomes is compared with critical point drying. The two procedures are equivalent for preparing chromosomes for examination by field emission scanning electron microscopy at low voltage.
During DNA repair studies, cells are occasionally kept on ice in order to suppress DNA repair. In the present studies cultivated human NC37 B-lymphoblasts were damaged by UV-A irradiation (365 nm) and DNA single strand breaks were detected at the single cell level with the alkaline comet assay in the temperature range from 4 degrees C to 44 degrees C. Single cell studies, in contrast to bulk experiments, allow to identify apoptotic or necrotic cells, which can be omitted for data analysis. Unexpectedly, similarly efficient single phase repair kinetics was found at all temperatures below 37 degrees C, i.e., particularly also in the cold. For recovery times below 20 min a linear decrease of DNA damage was detected. After 20 min, no additional repair was observed, i.e., complete repair of single strand breaks was not achieved. At 44 degrees C DNA damage increased with time, probably due to heat damage and cell death. Nucleotide excision repair inhibitors such as aphidicolin, 1-beta-D-arabinofuranosyl cytosine (araC) and hydroxyurea, but not the base excision repair inhibitor methoxyamine caused a strong increase in DNA strand breaks. The use of repair inhibitors confirmed DNA repair at 4 degrees C. In conclusion, partial repair of UV-A damage is similar at 37 degrees C and 4 degrees C and is probably governed by nucleotide excision repair. Keeping samples on ice may not result in a total suppression of DNA repair.
The parameter tail moment in single cell gel electrophoresis (comet assay) is calculated as the product of the two values: the percentage of DNA in the comet tail and the tail length in microm. Experiments were performed with cultured mammalian cells: B-Lymphoblasts, epithelial cells of a kidney tissue and a plate-epithelial cell line of a human carcinoma. They were irradiated in suspension with UV A at lambda = 343 nm, generated by an excimer laser-pumped dye laser. DNA migration was assessed and analysed. It is demonstrated that the distribution of the tail moments can be fitted by a chi2 (chi-square) distribution, whereas the factors of the product tail moment tend to be normally distributed. From this result, consequences for the statistical evaluation of the results can arise, especially for the computation of the confidence limits and for the valuation of the parameter tail moment from other comet assay experiments.
T2*-weighted imaging at 4.7 T was used to identify the cortical areas activated by electrical stimulation of the forepaw and hindpaw of alpha-chloralose anesthetized rats. Variation of the coronal slice position relative to the bregma, showed that the forepaw representation in the somatosensory cortex is more frontal and lateral than that of the hindpaw. Overlap between both activation areas was observed only in a small region in the slice at the level of the bregma. Documented localizations of both representations are in good agreement with earlier observations using invasive techniques. The determination of the separate areas of both paws indicates the feasibility of more complex activation studies in anesthetized animals, such as combined stimulations for the investigation of potentiation or depression effects on individual stimuli.
Functional activation of somatosensory cortex was studied in alpha-chloralose anesthetized rats by functional magnetic resonance imaging (fMRI), using both perfusion-weighted and T2*-weighted (blood oxygenation level dependent, BOLD) imaging. The sensitivity of functional activation was altered by ventilating animals for 3 minutes with 6% CO2. Before hypercapnic conditioning, electrical stimulation of the left forepaw at a frequency of 3 Hz led to an increase of signal intensity (relative to the unstimulated baseline condition) in the right somatosensory cortex by 6+/-2% (means+/-SD) in T2*-weighted images and by 45%+/-48% in perfusion-weighted images. After hypercapnic conditioning the signal intensity increase in perfusion-weighted images doubled to 91%+/-62% (P=0.034), whereas that of T2*-weighted images only marginally increased to 7+/-4% (not significant). This different behavior in both imaging modalities is interpreted as evidence for an increased flow response in combination with a higher oxygen extraction. Thus, the fMRI data reflect hypercapnia-induced resetting of the functional-metabolic coupling of the tissue during activation.
Recovery of the cerebral cortex after 10 min of cardiac arrest was studied in rat using noninvasive MRI techniques. The apparent diffusion coefficient (ADC) of brain water was imaged to document reversal of the metabolic impairment. Perfusion-weighted imaging and blood oxygen level dependent (BOLD) imaging were performed to assess functional recovery. To this purpose, rats were anesthetized with alpha-chloralose, and somatosensory cortex was activated by electrical stimulation of the contralateral forepaw. In sham-operated controls, cortical ADC was 862 +/- 10 microm2/s, and stimulation of forepaw led to a focal increase of signal intensity in somatosensory cortex by 71 +/- 22% in perfusion-weighted images and by 6 +/- 1% in BOLD images. One hour after successful resuscitation following 10 min of cardiac arrest, ADC did not differ from control but functional activation was completely suppressed. After 3 hours of reperfusion, functional activity began to reappear but the recovery of the BOLD signal progressed faster than that of the perfusion-weighted signal. The differences in the recovery of ADC, BOLD, and perfusion imaging are related to differences between metabolic and functional recovery on one hand and between blood flow and oxygen extraction on the other. The combination of these MRI methods thus provides detailed qualitative information about the progression of brain recovery after transient circulatory arrest.
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A fast version of the arterial spin tagging technique for the detection of cerebral perfusion is presented. Based on adiabatic spin inversion in combination with snapshot FLASH imaging, our technique allows the recording of perfusion changes with a temporal resolution of about 3 s. Differences of cerebral perfusion dependent on the choice of anesthesia were observed in rat brain. Furthermore, with this arterial spin tagging method we demonstrated perfusion increases in the somatosensory cortex of anaesthetized rats during forepaw stimulation. Comparison of the activated areas in the T2 -weighted BOLD images and the perfusion-weighted images showed good spatial correspondence, but the sensitivity to the functional activation was more than ten times higher in the perfusion technique.
The dependence of functional MRI contrast on the repetition rate (1.5-9 Hz) of a sensory stimulus was investigated with a T2*-weighted gradient echo method during forepaw stimulation of alpha-chloralose anesthetized rats (n = 5). An activation area was observed in the left or right somatosensory cortex dependent on the stimulation of the right or left forepaw, respectively. The activation intensity decreased for stimulation frequencies above 3 Hz, and was negligible at 9 Hz. The interpretation is that at low stimulation rates the neurons can respond to each stimulus, but at higher rates there is insufficient recovery time so that the response is progressively occluded.
Eight bacterial isolates from enrichment with 2,4,6-trichlorophenol (TCP) as sole carbon source were tested for their potential to degrade prochloraz. None of them could grow on prochloraz. Strain C964, identified as Aureobacterium sp., effectively reduced the fungitoxic activity of prochloraz in a bioassay and degradation was confirmed by HPLC. Two other isolates, strain C611 and C961, using TCP as a carbon source, belong to the beta subclass of the proteobacteria and presumely degrade TCP via 2,4-dichlorohydroquinone and hydroxyhydroquinone as indicated by oxygen-consumption tests.
The coordination between the trunk and arm of six subjects was examined during unrestrained pointing movements to five target locations. Two targets were within arm's length, three were beyond. The trunk participated in reaching primarily when the target could not be attained by arm and scapular motion. When the trunk did contribute to hand transport, its motion started simultaneously with arm movement and continued until target contact. Redundancy in the degrees of freedom used to execute the movement had no effect on the configuration of joints and segments used to attain a specified target; no difference in variability was noted regardless of whether redundancy existed. However, different configurations were used to achieve the same wrist coordinates along a common endpoint path, depending on the final position of the hand. The addition of trunk flexion, rotation and scapular motion did not alter the coupling between the elbow and shoulder joints and had no effect on the path of the hand or the smoothness of its velocity profile. Thus, trunk motion was integrated smoothly into the transport phase of the hand. As the trunk's contribution to hand transport increased, it played a progressively greater role in positioning the hand close to the target during the terminal stage of the reach. Of the movement components measured, trunk flexion was the last component to complete its motion when target reaches were made beyond arm's length. Hence, the trunk not only acts as a postural stabilizer during reaching, but becomes an integral component in positioning the hand close to the target.
The electron transfer in photosystem I (PS I) from the secondary acceptor A1 to the iron-sulfur centers is studied by X-band transient EPR with a time resolution of approximately 50 ns. Results are presented for a series of different PS I preparations from the cyanobacterium Synechococcus 6301 ranging from whole cells to core particles in which the iron-sulfur centers have been successively removed. In addition, results from PS I preparations from spinach and the cyanobacterium Synechocystis 6803 are presented. In all samples containing iron-sulfur centers, two consecutive spin-polarized EPR spectra are observed. The two signals have previously been assigned to the charge-separated states P700+.A1-. and P700+.(FeS)-, where (FeS) is one of the three iron-sulfur centers, FX, FA, or FB [Bock, C., van der Est, A., Brettel, K., & Stehlik, D. (1989) FEBS Lett. 247, 91-96]. In agreement with this, the second spectrum is not observed in the sample in which the iron-sulfur centers have been removed. For (P700-FX), core particles which do not contain FA and FB, the second spectrum can unambiguously be assigned to the pair P700+.FX-. In all samples containing FX, the transition from the first to the second spectrum occurs with t1/e approximately 280 ns (t1/2 approximately 190 ns) both in the presence and absence of FA and FB, which strongly suggests that this phase reflects electron transfer from A1-. to FX in intact PS I.(ABSTRACT TRUNCATED AT 250 WORDS)
Spin-polarized EPR spectra of the triplet state of P700, the primary electron donor in photosystem I (PS I), have been measured for the first time at room temperature. The measurements were performed on intact PS I from Synechococcus sp. after prereduction of all iron-sulfur centers and on vitamin K1 depleted PS I from Synechocystis 6803. The two preparations give similar spectra with a polarization pattern which indicates that the triplet state is formed via recombination of a radical pair. The axial and nonaxial zero-field splitting (zfs) parameters are found to be magnitude of D = (284 +/- 15) x 10(-4) cm-1 and magnitude of E = (22 +/- 3) x 10(-4) cm-1, respectively. The E-value is 42% smaller than in monomeric chlorophyll a, while the D-value is nearly the same. Measurements of the Synechocystis 6803 sample at 4.5 K yielded zfs parameters which are identical with those of the chlorophyll monomer, in agreement with previous results. In order to explain this behavior, it is proposed that the triplet excitation is delocalized over the two halves of a chlorophyll dimer at room temperature but appears localized on one half at low temperature. The observed zfs parameters are obtained if (1) the magnetic z-axes of the two chlorophylls are collinear, (2) the magnetic y-axes (and x-axes) of the two chlorophylls make an angle of approximately 55 degrees with each other, and (3) the admixture of charge-transfer states to 3P700 is negligible.(ABSTRACT TRUNCATED AT 250 WORDS)
Retinoic acid (RA) receptors (RARs) are ligand-inducible transcription factors that bind to specific DNA sequences associated with the regulatory regions of RA-regulatable genes. Since RA has been implicated as an important factor both in normal development and in teratological studies, one would like to have a model system that detects the presence of activated receptors during development. We have constructed a recombinant reporter gene that has three copies of the RA response element (RARE) from the RAR beta-2 promoter 5' to the herpes simplex virus thymidine kinase promoter; this regulatory region is coupled to the bacterial beta-galactosidase reporter gene. This construct was RA inducible in transient transfection assays in F9 embryonal carcinoma cells. Transgenic embryos with this reporter gene construct exhibited restricted and reproducible patterns of beta-galactosidase activity during embryogenesis, beginning between gestational ages day 7.5 and 8.5. At day 8.5, beta-galactosidase activity was detected in the closed neurotube and somites. Day 8.5 embryos, from pregnant females fed RA 14 hr earlier, exhibited a greater intensity and distribution of beta-galactosidase activity. Similarly, at later stages of gestation, maternal RA exposure resulted in enhanced embryonic beta-galactosidase expression. This type of transgenic indicator mouse should be useful in detailing the role of activated RARs during embryonic development.
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Rectal cancer patients are at high risk for disease progression even after radical surgery for cure. Prognosis depends on a timely diagnosis for effective, curative therapies. In resected patients, an accurate follow-up especially within the first two years of surgery, is required. Clinical and radiologic follow-up is based on periodical, timed physical and instrumental exams (rectal exploration, neoplastic markers, rectoscopy, colonic enema, CT MRI, TRUS) which supply information on locoregional (liver, lymph nodes, peritoneum, chest) recurrence. Rectal exploration, endoscopy, colonic enema and TRUS enable the control of anastomosis and the identification of possible metachronous tumors. CT and MRI as liver US and chest X-ray, detect recurrence in other sites (pelvic perianastomotic recurrence, peritoneal carcinomatosis, lymph node or liver locations). A diagnostic problem is represented by the difficult differentiation of local recurrence from postoperative fibrosis. In this case, surveillance in the course of time with exams more reliable in tissue differentiation (MRI, though with some limitations) and US- or CT-guided biopsy, is required.
INTRODUCTION: In the last 25 years, random trials on the advantages of combined surgery and irradiation/chemotherapy of breast cancer demonstrated similar survival rates to those of massive surgery. However, both irradiation and chemotherapy have advanced and now yield good local control of the disease, so that even large breast cancers can be made operable. Breast cancer follow-up is carried out with imaging and clinical examinations to detect early locoregional recurrences, contralateral lesions and distant recurrences: to this purpose, we carried out a comparative study of all imaging modalities. MATERIAL AND METHODS: We retrospectively reviewed the data of 42 breast cancer (T2-T3 N0-N+) patients of the Senology Center of the Catholic University (Rome, Italy) treated with irradiation and/or chemotherapy for tumor debulking to permit conservative surgery. We investigated the sensitivity and the indications of mammography, B-mode and color Doppler US and MRI in measuring the exact tumor size and detecting locoregional metastatic nodes. RESULTS: After 3 years' treatment, our recurrence rate (19%) is a little higher than those in the major international trials (4.2-9% and 5 tears). The recurrence was on the surgical scar in 75% of cases (6/8), while multifocal tumors were found in 25% of cases. US was the most accurate method in measuring tumor size in 90% of cases (18/20), while mammography frequently overstaged the lesion and yielded exact measurements in 65% of cases (13/20). MRI was as accurate as US, but this technique is too expensive and little available in Italy. US accurately diagnosed lymph node recurrences (70% sensitivity), but MR rate was even higher (80%), while mammography and color Doppler US had only 5-10%. As for treatment outcome, an irregular and blurred nodule or multifocal lesions at mammography indicate poor/no response, while a much smaller radial scar than at previous similar follow-ups indicates treatment success. CONCLUSIONS: When correctly integrated, mammographic, US and MR patterns permit exact tumor size measurement and show possible locoregional lymph node involvement in the patients submitted to conservative surgery and irradiation/chemotherapy. In contrast, color Doppler findings remain poorly specific in this disease, with about 55% sensitivity. Therefore, radiologic studies, with clinical and laboratory data, have a major prognostic value in assessing the biological response to combined treatment.
INTRODUCTION: Gynecomasty is usually classified as normal of abnormal, except for 25% of cases which are classified as idiopathic because their causes and pathogenesis remain unknown. Gynecomasty is diagnosed mainly on clinical grounds, while integrated imaging, sometimes combined with cytology, is used to distinguish benign from malignant forms. Bilateral gynecomasty is easy to diagnose, especially when patients report assuming particular drugs or present other risk factors, but unilateral or asymmetrical gynecomasty is a diagnostic problem. Primary male breast cancer usually presents as a unilateral hard mass, often infiltrating the dermis and with early lymph node metastases; it is associated with gynecomasty in 20% of cases. MATERIAL AND METHODS: We examined 76 men (age range, 15-75 years) referred for breast enlargement; the patients with radiologic findings of breast adiposis were not included in our series. All patients were submitted to standard projection mammography with a high resolution dedicated film and to real time US with high frequency probes (7.5-12 MHz). RESULTS: Breast enlargement was unilateral in 48% of cases and bilateral in 52%. The radiologic patterns, compared with histologic or clinical-therapeutic follow-up, permitted the correct diagnosis in 72 of 76 patients (94%). The extant four patients had chronic inflammation (3 cases) and a malignant tumor with questionable imaging features. Overall imaging findings were: 55 cases (72%) of actual gynecomasty--unilateral in 17 and bilateral in 38 cases--9 unilateral malignant tumors (12%), eleven cases of inflammation (14%) and 1 case (2%) of unilateral metastasis from plasmocytoma. Sixteen (29%) actual gynecomasty patients (21% of the whole series) had a nodular form (unilateral in 6 and bilateral in 10 cases), 23 (42% and 30% of the whole series) had a dendritic form (unilateral in 7 and bilateral in 16 cases) and 16 (29%, 21% of the whole series) had a glandular form. CONCLUSIONS: Diagnosing gynecomasty is relatively easy in the patients with bilateral forms with a positive history of associated exogenous or endogenous factors, while focal unilateral or asymmetrical forms are difficult to distinguish into benign and malignant. The radiologic pattern may be questionable especially in chronic inflammation and in some malignant forms and must therefore be integrated with cytologic or surgical findings.