ALT-EJ genera rearreglos cromosómicos en respuesta a Etopósido en células humanas con los principales sistemas de reparación de rupturas de doble cadena comprometidos / ALT-EJ originates chromosomal rearrangements in response to Etoposide in human cells with the main DNA double-strand break repair systems compromised

RESUMEN La droga antitumoral Etopósido (ETO) induce rupturas de doble cadena en el ADN (RDC) y promueve el desarrollo de neoplasias secundarias en los pacientes tratados. Dos mecanismos principales, recombinación homóloga (HR) y reunión de extremos no-homólogos clásica (c-NHEJ) reparan las RDC. Cuando HR y c-NHEJ son defectuosas, la vía alternativa de reunión de extremos (alt-EJ) dependiente de PARP-1 está implicada. Se examinó la participación de alt-EJ en la progresión de las RDC inducidas por ETO en la fase G2 de células humanas. Se establecieron células HeLa deficientes en HR (inhibición de cohesina RAD21, HeLa RAD21kd) y su control no-silenciada (HeLa NS). Las células se trataron con ETO en presencia del inhibidor químico de DNA-PKcs (DNA-PKi, c-NHEJ). En ambas líneas celulares, la inducción de RDC (γH2AX+) por ETO en la fase G2 aumentó respecto a sus controles. La reparación incorrecta en células deficientes en DNA-PKcs y RAD21 originó un incremento sinérgico de intercambio de cromátidas y de cromosomas dicéntricos en la primera y segunda metafase, respectivamente. En cambio, la frecuencia de cromosomas dicéntricos se redujo en células deficientes en PARP-1 (HeLa PARP- 1kd) luego del tratamiento con ETO. En células binucleadas HeLa RAD21kd, DNA-PKi/ETO acrecentó el porcentaje de células con ≥20 focos γH2AX en la fase G1-posmitótica y de micronúcleos a las 96 h. Una mayor acumulación en G2/M se observó en HeLa NS tratadas con DNA-PKi/ETO en relación a HeLa RAD21kd a las 8 h. El ciclo celular se reanudó en HeLa NS a las 16 h, sin embargo, la acumulación se mantuvo en HeLa RAD21kd. Los rearreglos cromosómicos obtenidos con DNA-PKcs y RAD21 disfuncionales y su disminución en células HeLa PARP-1kd, sugieren que alt-EJ contribuye a su formación.

ABSTRACT The antitumor drug Etoposide (ETO) induces DNA double-strand breaks (DSB) and is associated with the development of secondary neoplasms in treated patients. DSB are repaired by two main mechanisms, homologous recombination (HR) and classical non-homologous end joining (c-NHEJ). When HR and c-NHEJ are defective, DSB are repaired by the PARP-1-dependent alternative end-joining (alt-EJ) pathway. The involvement of alt-EJ in the progression of DSB induced by ETO in the G2 phase of human cells was analyzed. HeLa cells deficient in HR (cohesin RAD21 inhibition, HeLa RAD21kd) and their nonsilencing control (HeLa NS) were established. Cells were treated with ETO in the presence of a chemical inhibitor of DNA-PKcs (DNA-PKi, c-NHEJ). In both cell lines, ETO-induced DSB (γH2AX+) in G2 phase were increased compared to their controls. The incorrect repair of DSB in DNA-PKcs- and RAD21-deficient cells caused a synergistic augment in chromatid exchanges and dicentric chromosomes in the first and second metaphase, respectively. In contrast, the frequency of dicentric chromosomes was reduced in PARP-1-deficient cells (HeLa PARP-1kd) following ETO treatment. In HeLa RAD21kd binucleated cells, DNA-PKi/ETO increased the percentage of cells with ≥20 γH2AX foci in the G1-postmitotic phase and of micronuclei at 96 h. A greater accumulation in G2/M was observed in HeLa NS treated with DNA-PKi/ ETO compared with HeLa RAD21kd at 8 h. The cell cycle restarted in HeLa NS at 16 h; however, the G2/M accumulation was maintained in HeLa RAD21kd. Chromosomal rearrangements obtained when DNAPKcs and RAD21 were absent and their decrease in HeLa PARP-1kd cells suggest that alt-EJ contributes to their formation.

Spring constant calibration of atomic force microscopy cantilevers with a piezosensor transfer standard.

We describe a method to calibrate the spring constants of cantilevers for atomic force microscopy (AFM). The method makes use of a "piezosensor" composed of a piezoresistive cantilever and accompanying electronics. The piezosensor was calibrated before use with an absolute force standard, the NIST electrostatic force balance (EFB). In this way, the piezosensor acts as a force transfer standard traceable to the International System of Units. Seven single-crystal silicon cantilevers with rectangular geometries and nominal spring constants from 0.2 to 40 Nm were measured with the piezosensor method. The values obtained for the spring constant were compared to measurements by four other techniques: the thermal noise method, the Sader method, force loading by a calibrated nanoindentation load cell, and direct calibration by force loading with the EFB. Results from different methods for the same cantilever were generally in agreement, but differed by up to 300% from nominal values. When used properly, the piezosensor approach provides spring-constant values that are accurate to +/-10% or better. Methods such as this will improve the ability to extract quantitative information from AFM methods.

Ruptured abdominal aortic aneurysm: role of initial delay on survival.

In the years 1990 to 1997, 103 patients with RAAA were operated on at the Department of Vascular Surgery of the Hospital Na Homolce in Prague. Men outnumbered women, mean age was 70 years. The mean delay between onset of symptoms and hospital admission was 25 hours. Prior to transportation 85 patients were submitted to at least one confirmative evaluation test (CAT, ultrasound, angiography) and 33 patients to a combination of two or more herementioned examinations. Twenty-eight patients were referred via two or more hospital departments. In 71% of patients profound shock with oligoanuria and hypotension was found upon admission. Anuria/hypotension proved to occur in a significantly lower rate in later survivors compared to later non-survivors (S vs. NS = 30% vs. 92.1%, p < 0.002) and preoperative hematocrite and S-creatinine values copied the clinical trend. At surgery, persistent hypotension together with necessity of resuscitation steps as well as finding of free blood within the abdominal cavity showed up as further significant death predictors. Postoperatively, acute renal and/or multiorgan failure occurred in 36 patients and significantly prevailed in the NS vs. S group (48.3% vs. 22.5%, p < 0.03). Both early hemorrhage and myocardial infarction infavorably influenced the outcome. Seven patients (6.8%) expired during operation. The total of 63 patients died (61.2%) fifty-eight patients within the 30-day period (56.3%). Within the first five days 58.7% of all deaths occurred particularly related to hemorrhagic shock. The latter fatalities (41.3%) were caused by both organ failure and septic complications. In our cohort regardless of age, type or extent of surgery, outcome was determined by status upon admission. Delay in surgical treatment caused both by time consuming confirmative evaluation and patient's lengthy transfers is responsible for ominous protraction of the original shock. Especially in intraperitoneal rupture, the irreversible sequels of devastating hemorrhage only rarely do not lead to a fatal end albeit the patients survive the aortic reconstruction.

[Diagnostic significance of mild hyperhomocysteinemia in a population of children with parents or grandparents who have peripheral or coronary artery disease]. / Diagnostický význam mírné hyperhomocysteinémie v populaci detí narozených z rodicu ci prarodicu trpících onemocnením periferních ci koronárních tepen.

BACKGROUND: A rise of the homocysteine plasma level--mild hyperhomocysteinaemia--is considered an independent risk factor for the development of vascular damage. It is due to hereditary deficiency of 5,10-methylene-tetrahydrofolate reductase with accentuation of vitamin deficiency (folic acid, vitamin B6 and B12). In previous studies the authors confirmed this fact in the population of patients with aortocoronary or peripheral arterial bypasses. The assumed autosomal recessive transmission of this deficiency should make it possible to detect carriers of this metabolic deviation already in childhood. By selective screening of the child population at risk it would thus be possible to detect affected subjects in time and prevent the development of vascular disease by preventive folate administration. METHODS AND RESULTS: In a group of 38 children and grandchildren from risk families where at least one of the parents or grandparents was operated on account of vascular obliterating disease the total homocysteine plasma level was examined by the chromatographic method. An increase of total homocystein (8.7 +/- 2.7 mumol/l) was found as compared with children from the non-risk population (5.4 +/- 1.8 mumol/l), (p < 0.001). The total homocysteine values however were dependent on the child's age and were more marked in children above 12 years of age. In the parental population mild hyperhomocysteinaemia was present in 38% of those with aortocoronary bypasses and in 43% of those with peripheral arterial bypasses. CONCLUSIONS: The authors found significantly elevated total homocysteine levels in the child population from risk families with obliterating vascular disease. The total homocysteine level depends on the child's age and is more markedly expressed in children above 12 years of age.

[Hyperhomocysteinemia. A risk factor for the development of vascular diseases not associated with lipid levels]. / Hyperhomocysteinémie. Rizikový faktor pro vznik cévních onemocnení nezávislý na hladinách lipidu.

BACKGROUND: Elevated total homocysteine plasma levels are considered a significant factor of vascular damage. As they are encountered in more than half the patients with atherosclerotic vascular damage the importance as a lipid-dependent or lipid-independent risk factor in the promotion of pathophysiological processes is discussed. METHODS AND RESULTS: In a group of 100 healthy subjects and 529 patients with indication for an aortocoronary or peripheral arterial bypass and in patients from the lipid clinic the mutual relation between total plasma homocysteine levels and selected indicators of the lipid metabolism was investigated. The following results more obtained: for total cholesterol a correlation coefficient of r = 0.26, for HDL-cholesterol r = 0.20, for LDL-cholesterol r = 0.21, for triacylglycerols r = 0.29, apolipoprotein A-I r = 0.06, apolipoprotein B r = -0.12 and for Lp(a) r = -0.03. To ensure correct evaluation of the homocysteine levels simultaneously also folate levels were examined (correlation coefficient r = 0.28), vitamin B12 r = (0.03) and fibrinogen r = (0.09). CONCLUSIONS: The authors did not detect an unequovical relationship between the total homocysteine level and selected lipid indicators in any of the patient groups (p < 0.05).

[Ruptured abdominal aortic aneurysm]. / Aneurysma aortae abdominalis cruens.

In the years 1990-1994, 43 patients with ruptured abdominal aortic aneurysms (RAAA) were operated on at the Department of Vascular Surgery of the Na Homolce Hospital in Prague. Men outnumbered women, average patient age was 70 years. The mean delay between onset of symptoms and hospital admission counted 27 hrs. Prior to transportation, one half to two thirds of patients went through at least two types of confirmative evaluation (CAT, ultrasound, angiography) and/or were referred via two or more hospital departments. In two thirds of patients profound shock with oligoanuria and hypotension were found. Anuria/hypotension proved to occur in a significantly lower rate in later survivors compared to later dead (11.8% vs. 23.5%: p < 0.05). Persistent hypotension during surgery together with eventual resuscitation as well as free blood found within the abdominal cavity showed up as further ominous factors. Renal failure was the leading postoperative complication (51.2%) with 27.9% of patients requiring hemodialysis after repair. Sepsis (25.6%), pneumonia (20.9%) and hemorrhage (13.9%) followed. Twenty-six patients were lost (60.5%) either within the first hours and days after surgery because of irreversible hemorrhagic shock or between the second and fourth week due to the sequels of organ failure and sepsis. In our cohort, regardless of age, sex, concomitant disease or the type of surgery, the patient's status on admission determined his/her further destiny. Urgent transfer to a specialized center going hand in hand with prompt and effective reanimation steps are the patient's only hope for survival.

Pharmacodynamic study of the capillaries of normal and allergic (atopic) subjects.

Using the capillary resistance test as a method to study the cutaneous microvessels, a number of capillary active agents were investigated by applying them percutaneously in hydrophylic ointment base to manifestly allergic and definitely non-allergic volunteers. While the catecholamines and heparin increased capillary resistance in both allergic and non-allergic groups, metacholine, serotonin, the somatotropic hormone and nitroglycerin either did not change or decreased capillary resistance in the normal group and invariably increased it in the members of the allergic group. The effects of catecholamines were, completely or partially, blocked in the normal group by pretreatment of the skin with dibenzyline or propranolol. In the members of the allergic group, blockade was often weak or absent indicating some derangement in the adrenergic receptors. The significance of the findings is discussed.